UC-NRLF 


SB 


» 


» 


•? 


w  -/ 

l  /<•  I-  ¥  0 


<S%- 

-V  -^ 


a 

A 


^:^l 


COMPARATIVE  VIEW  OF  THE  PRINCIPAL  BACTERIA  OF  DISEASE.      (AFTER  CORNIL  AND  HAKES.) 

XI035. 


REFERENCES    TO    PLATE    XIO35. 


1.  Anthrax,  a.  b.  c  d  and  a.  b.  c.  d. 

e.  f  gr.h. 

2.  Chicken  Cholera,  a.  b  c. 

3.  Glanders,  a  b  c, 

4.  Typhoid  Fever,  a.  b. 

5.  Erysipelas, 

6.  Puerperal  Fever. 

7.  Yellow  Fever,  a.  b. 
8    Measles,  a  b.  c 

q.  Diphtheria,  a.  b.  c.  d. 

10.  Pneumonia. 

11.  Hog  Cholera,  a.  b. 

12.  Variola. 

13.  Vac>.«  la. 

14.  Noma. 

15.  Gangrene,  a.  b. 


16.  Gonorrhoea,  a  b. 

17.  Septicaemia  of  Mice,  with  Kid- 

ney Lesions. 

18.  Pyaemia. 

19.  Endo-carditis. 

20.  Parenchymatous  Nephritis. 

21.  Cerebro  spinal  Meningitis. 

22.  Tuberculosis,  a  b.  c. 

23.  Tuberculosis,  d.  e. 

24.  Leprosy,  a.  b.  c.  d . 

25.  Whitlow. 

26.  Red  S  \veat. 

27.  Wart. 

28.  Jequirity,  a.  b.  c.  d.  e.  f.  g.  h.  i, 

29.  Recurrent  Fever. 


AN   INTRODUCTION 


PRACTICAL    BACTERIOLOGY. 


A    GUIDE    FOR 

STUDENTS    AND     GENERAL     PRACTITIONERS. 


THOMAS  E.  SATTERTHWAITE,   M.   D., 
M 

Professor   of  Pathology   and    General  Medicine   in  the   New    York 

Post    Graduate    Medical   School   and   Hospital, 

New     York    Citv. 


1887. 
GEORGE  S.  DAVIS, 

DETROIT,   MICH. 


..3-3 


Copyrighted  by 

GEORGE  S.   DAVIS, 

1887. 


TABLE  OF  CONTENTS. 


iAPTER  PAGE. 

I.     Introductory , i 

II.     The    Microscope    and    Microscopical   Appliances 

for  Use  in  a  Bacteriological  Laboratory     ...  13 

III.  Methods  of  Examination 43 

IV.  Culture  Methods 51 

V.     Methods  of  Investigating  Special  Bacteria 60 


BOOKS  OF  REFERENCE: 


Les  Bacteries.     Cornil  &  Babes.     Paris,  1885. 

Micro- Organisms  and  Diseases.     Klein.     London,  1885. 

Pathological  Mycology.     Woodhead  &  Hare.     Edinburgh, 

1885. 

•v 

Die  Micro-organismen.     Fluegge.     Leipsig,  1886. 
Practical  Bacteriology.    Crookshank.    New  York,  1886. 
Bacteriologische     Untersuchungs-Methode.        Huber     & 
Becker.     Leipsig,  1886. 


PREFACE. 


It  has  been  the  aim  of  the  writer  in  issuing  this  little 
monograph  to  furnish  the  student  and  medical  practitione 
with  a  concise  resume  of  bacteriology,  practical  in  character, 
and  so  extend  more  widely  an  interest  in  this  most  important 
topic. 

A" single  chapter  has  been  devoted  to  the  subject  of 
Germ  Theories,  and  to  the  successive  advances  that  have 
been  made  towards  securing  our  present  knowledge. 

Bacteriology,  as  a  branch  of  medicine,  has  already  ob- 
tained for  itself  a  name  and  permanent  place,  in  spite  of  the 
many  obstacles  and  the  vigorous  opposition  it  has  encount- 
ered; and  yet  it  will  be  conceded  by  the  most  ardent  bacte- 
riologist that  many  of  its  fundamental  principles  are  shrouded 
in  obscurity. 

But  there  is  good  reason  to  believe  that  the  researches 
of  the  next  few  years  will  yield  rich  results,  for  steady  pro- 
gress is  now  being  made  towards  the  perfection  of  those 
instruments  of  precision  that  are  necessary  for  future  work, 
and  we  may  confidently  hope  that  the  morphology  of  these 
microphytes,  their  proper  classification,  chemical  qualities 
and  physiological  attributes  will  soon  be  satisfactorily  estab- 
lished. 

As  the  scope  of  this  little  guide  precluded  much  origin- 
ality, the  writer  has  made  no  special  efforts  in  that  direction, 
but  has  freely  borrowed  from  the  subjoined  list  of  works, 
which  may  be  consulted  for  many  details  that  were  inad- 
missible for  manifest  reasons.  Thanks  are  here  given  to 
Dr.  J.  M.  Rice  for  his  assistance  in  preparing  the  chapters 
on  methods,  general  and  special;  also  to  Messrs  Eimer  & 
Amend,  of  this  city,  for  the  use  of  many  electro-plates. 

T.  E.  S. 

17  E  44th  Street,  New  York  City,  April  20,  1887. 


CHAPTER  I. 

I-NTRODUCTORY. 

As  prefatory  to  this  subject,  I  think  it  advisable 
to  reproduce  some  of  the  points  of  a  paper  read  in 
Philadelphia,  before  the  International  Medical  Con- 
gress, of  1876,  partly  because  certain  conclusions 
were  there  maintained  in  the  section  on  sanitary  sci- 
ence, before  which  the  topic  was  discussed,  and  be- 
cause they  were  further  sustained  by  the  Congress 
sitting  as  a  whole;  and  also  for  the  reason  that  they 
will  serve  as  a  fitting  introduction  to  the  general  sub- 
ject. And  it  may  be  remembered,  incidentally,  that  at 
that  remarkable  gathering  there  were  present  some  of 
the  most  prominent  advocates*  of  what  was  then  called 
the  germ-theory.  In  enumerating  at  that  time  the 
various  theories  and  hypotheses  that  had  been  framed 
to  explain  the  origin  of  infective  diseases,  three  were 
given,  viz.:  (i)  the  vegetable  germ;  (2)  the  bioplasm; 
(3)  the  physico-chemical,  and  they  were  briefly 
sketched  as  follows: 

I.  It  was  said  that  the  vegetable-germ  theory 
had  attracted  most  attention,  but  was  a  flimsy  hypo- 
thesis until  the  microscope  came  into  use.  Then  it 
was  that  Schwann,  Cagniard  de  Latour  and  Ktitztngt 


*  Lister,  Hueter. 

f  Schutzenberger,  on  Fermci  tations,  pp.  36-7. 


2     

observed  that  there  was  a  growth  of  microscopic 
organisms  in  fermenting  fluids,  and  when  at  a  later 
date  Pasteur  maintained  that  these  organisms  actually 
produced  the  fermentative  process,  then  medical 
men  inquired  whether  it  might  not  be  true  that  in- 
fective diseases  had  an  analogous  origin.  But  the  best 
a  priori  evidence  of  the  truth  such  theories  presented 
was  adduced  by  Schonlein,  who,  in  1838,  published  his 
account  of  the  microscopic  organism  found  in  favus, 
or  honeycomb  ring-worm.  And  he  substantiated 
his  views  in  so  able  a  manner  that  the  plant  has  come 
to  be  held  by  dermatologists  and  others  as  the  cause 
of  the  disease.  It  is  classified  under  the  moulds 
or  fungi.  At  the  time  when  the  paper  before  alluded 
to  was  read,  very  many  of  the  infective  diseases  had 
been  alleged  to  have  a  specific  microphyte,  causing 
the  disease  by  their  own  inherent  poisonous  qualities. 
Such,  for  example,  was  the  measles-fungus  of  Salis- 
bury, the  diphtheria  micrococcus  of  Letzerich,  the 
cholera-fungus  of  Hallier,  described  in  1867,  the 
microphaerae  vacciniae  of  Cohn,  the  bacteridiae  of 
anthrax,  discovered  by  Pollender  and  Davaine,  the 
spirillus  of  relapsing  fever,  discovered  by  Obermeier, 
and  the  microcytes  of  typhoid  fever,  described  by 
Klein,  with  some  others  whose  claims  to  recognition 
were  less  definite. 

II.     The  second  hypothesis  was  that  which  prob- 


f  Bastian,  Lancet,  April  10,  1875,  p.  502. 


—  3  — 

ably  originated  with  Beale.*  He  was  willing  to  ad- 
mit that  particles  of  microscopic  size,  molecules  in 
fact,  would  produce  disease,  but  he  held  that  they 
were  degraded  portions  of  the  animal  system.  And 
yet  they  had  the  power  of  dividing  and  subdividing 
under  diseased  conditions,  "as  living  matters  alone 
divide,"  to  quote  his  language.  The  distinguished 
Hutchinson,  of  London,  held  the  same  view,  believ- 
ing, in  particular,  that  gonorrhoea  and  purulent  oph- 
thalmia, erysipelas  and  phagedsena  were  communi- 
cated by  such  living  material. 

II.  The  third  hypothesis  was  well  expressed  at 
that  time  by  Bastian.  It  was  called  the  "  physico- 
chemical,"  or  "physical"  theory.  As  then  enunci- 
ated, it  held  that  though  minute  organisms  might 
act  as  ferments,  they  did  so  by  virtue  of  chemical 
actions  set  up  by  them,  while  minute  particles  of  the 
human  body  had  an  almost  equal  capacity  for  setting 
up  diseased  action  under  suitable  conditions.  In 
reference  to  the  relation  between  organisms  and  fer- 
mentation, I  must  be  permitted  to  quote  from  my 
own  paper,  which  states: 

"  After  the  publication  of  Pasteur's  brilliant  ex- 
periments in  relation  to  fermentation  and  putrefaction, 
they  were  regarded  as  affording  good  b  priori  evidence 
of  the  truth  of  the  doctrine  now  under  consideration. 
But  it  must  be  remembered  that,  though  sustained  by 


*  Beale,  Disease  Germs,  pp.  5  and  II. 


A         

the  observations  of  many  others,  these  views  were 
strenuously  opposed  by  Willis,  Stahl,  Liebig,  and 
others,  chiefly  of  the  German  school.*  There  is  little 
doubt  now  that  these  latter  were  in  a  measure  correct; 
in  fact,  Pasteurf  has  seen  fit  to  modify  some  of  his 
earlier  statements,  for  he  quite  recently  has  said  that 
both  alcoholic  fermentation  and  putrefaction  may  be 
initiated  by  the  chemical  processes  taking  place  in  the 
tissue-elements  of  certain  fruits  and  vegetables,  inde- 
pendently of  the  minute  organisms  supposed  to  be 
necessary  to  the  process.  Similar  statements  had  pre- 
viously been  made  by  MM.  Le  Chartier  and  Bellamy, 
who  found  that,  in  modified  forms  of  fermentation, 
independent  organisms  were  generally  absent  at  first, 
though  they  often  made  their  appearance  afterwards." 

In  a  subsequent  public  discussion  at  the  same 
congress,  in  another  section,  I  maintained  this  same 
ground,!  and  showed  that  there  had  been  and  were 
those  who  believe  that  fermentation  is  not  always  de- 
pendent on  the  growth  and  multiplication  of  living 
organisims.§ 

In  a  recent  and  most  valuable  article  in  which 
this  topic  comes  up  for  consideration,!  Prof.  Knapp, 

*  Schutzenberger,  p.  40. 

f  Lancet,  April  10,  1875,  p.  508,  and  Tribune  Med.,  April 
i,  1875,  P-  321. 

\  Trans,  of  the  Internal.  Med.  Congress,  1876,  p.  544. 

§  Handw.  d.  r.  u.  a.  China.  M.,  1848,  p.  232,  Proceedings 
of  the  Royal  Soc.,  No.  172,  1876. 

|  The  N.  Y.  Med.  Rec.,   Dec.  25,  1886,  p.  701. 


of  this  city,  cites  very  excellent  evidence  to  substanti- 
ate the  view  that  fermentation  is  caused  by  vegetable 
organisms,  and  it  is  proper  to  state  here  that  most 
chemists  do  rank  themselves  on  his  side,  which  is,  in 
fact,  the  prevailing  and  popular  one.  Prof.  Knapp 
alludes  especially  to  the  experiments  of  Schwann, 
Helmholz,  Schroeder,  and  von  Dusch.  And  he  says, 
"  the  microscope  has  shown  that  all  fermenting  sub- 
stances contain  yeast  plants,  and  experiments  in  vari- 
ous ways  have  demonstrated  that  no  fermentation 
takes  place  so  long  as  the  fermentative  substance  is 
kept  free  from  yeast  cells.  The  presence  of  microbes 
thus  being  an  essential  factor  in  fermentation;  the  de- 
finition of  fermentation  ought  to  express  it;  we  may, 
therefore,  define  fermentation  as  the  decomposition  of 
carbo-hydrates  into  simple  compounds  by  the  agency 
of  living  microbes." 

But  even  admitting  for  the  moment  that  some 
fermentations  are  so  produced,  these  statements  are 
far  too  sweeping;  that  is,  if  I  understand  rightly  the 
phraseology  used  by  some  of  our  best  and  latest 
writers;  thus  Sedgwick*  says,  "there  is  another  group 
of  obscure  chemical  phenomena  known  as  fermenta- 
tions, produced  by  certain  lifeless  or  inorganized  sub- 
stances." Such  are  the  mineral  acids,  sulphuric  or 
hydrochloric,  which  may,  under  certain  circumstances, 
decompose  starch,  cane  sugar,  and  some  other  organic 
substances  into  such  compounds,  without  themselves 

*  Reference  Handbook  of  the  Med.  Sci.,  1886,  III.  p.  63. 


undergoing  any  appreciable  changes.  Thus  cane  sugar 
is  inverted,  that  is,  turned  into  dextrine  and  glucose. 
Pepsine  is  another  example;  so  is  the  diastase  of  malt. 
And  it  is  a  matter  of  interest  that  the  conversion  of  cane 
sugar  into  grape  sugar  is  done  not  by  the  yeast  di- 
rectly, but  indirectly  by  a  soluble  ferment,  which  may 
be  extracted  from  the  yeast  and  will  act  in  its  ab- 
sence. Similar  statements  may  be  found  elsewhere, 
and  in  particular,  Prof.  Dittmar,  of  Glascow,  describes, 
under  the  fermentative  agents  proved  to  be  of  purely 
chemical  nature,  not  only  certain  acids,  diastase,  emul- 
sine,  peptine  and  pancreatine,  but  erythryzone,  a 
peculiar  ferment  discovered  by  E.  Schunck,  in  1854, 
and  extracted  from  madder  root,  and  which  he  found 
had  the  power  of  inducing  vinous  fermentation  in  solu- 
tions of  sugar.  All  of  these  ferments,  the  acids  ex- 
cepted,  lose  their  efficacy  at  a  temperature  of  about 
100°  C.,  in  the  presence  of  water.f 

I  stated  in  1876  that  DougallJ  had  produced  pu- 
trefaction without  bacteria,  and  that  Hiller§  made 
statements  to  the  same  effect,  for,  having  injected 
fresh  eggs  with  a  fluid  containing  bacteria,  but  not 
putrid,  the  eggs  remained  unaffected,  which  showed 
that  bacteria  might  be  present  without  decomposition 
ensuing.  These  views  received  further  support  from 
Donne  and  Beauchamp.  || 

f  Article  on  Fermentation,  in  Encyclo.  Brit.,  No.  7,  1879.. 
|  Dougall,  Brit.  Med.  Jour.,  Apr.  24,  1875,  p.  557. 
§  Centralbl.  f.  d.  med.  Wiss.,  Dec.,  1874. 
|  Schutzenberger,  p.  225. 


Now  we  are  told*  that  the  action  of  living  bac- 
teria is  as  essential  for  putrefaction  as  for  fermentation, 
and  that  it  can  be  prevented  by  adopting  proper 
methods,  such  as  the  exclusion  of  air,  washing  it, 
filtering  it,  or  by  gravitation  according  to  the  methods 
of  Pasteur  and  Tyndall.  Nor  is  this  all,  for  we  are 
toldf  that  putrefaction  and  suppuration  are  identical, 
since  it  is  now  alleged  that  the  bacteria  of  putrefac- 
tion will  also  produce  suppuration.  But  if  such  be 
the  case,  is  suppuration  always  due  to  a  parasite?  I 
will  quote  Dr.  Knapp's  statements  on  the  question: 
Does  traumatism  of  any  kind  produce  suppuration  ? 
"  I  made  a  series  of  experiments  in  Berlin  last  winter. 
I  performed  all  the  operations  that  are  practiced  on 
the  eye,  on  the  one  side  of  a  rabbit,  with  sterilized  in- 
struments, in  an  aseptic  way;  on  the  other  side  the 
wound  was  contaminated  with  an  emulsion  of  a  pure 
culture  of  some  pyogenic  fungus.  All  the  former 
healed  by  first  intention;  the  latter  suppurated  with 
the  regularity  of  a  chemical  experiment.  The  coarsest 
operation,"  he  continues,  "  the  rudest  treatment  of  a 
wound,  will  not  be  followed  by  suppuration,  if  only 
the  pyogenic  germs  are  excluded." 

As  to  the  question  whether  chemical  agents  pro- 
duce suppuration  without  the  intervention  of  microbes  ? 
Until  recently  we  are  told  that  opinions  were  divided 
on  this  subject,  but  that  latterly  the  experiments  of 

*  Knapp   loc.  cit. 
f  Knapp,  loc.  cit. 


Straus,  Scheuerlein,  Klemperer  and  Ruys  have  demon- 
strated to  almost  a  certainty,  that  bacteria  are  the 
causes  of  any  form  of  suppuration,  and  yet  Prof.  Knapp 
tells  us  that  many  of  the  most  competent  bacteriologists 
are  unwilling  to  admit  this  statement,  though  he 
firmly  believes  that  sufficient  evidence  has  been  ad- 
duced to  show  that  suppuration  in  any  case  depends 
on  the  action  of  microbes. 

I  will  now  introduce  the  conclusions  adopted 
after  the  reading  of  my  paper,  in  1876,  at  the  Congress 
in  Philadelphia: 

I.  That,  as  far  as  inquiry  has  been  made  as  to 
the  nature  of  the   active   principles  in  infective  dis- 
eases, it    is    probable   that  in  a  certain    number   the 
matter  is  particulate,  or  molecular  in  form. 

II.  That  in  regard  to  the  causes  of  septicaemia, 
pyaemia,  puerperal  fever,  erysipelas,  and  hospital  gan- 
grene,  and    those    of    cholera,   vaccine-disease,   the 
carbuncular  diseases  of  men  and  animals,  typhoid  and 
relapsing  fevers,  and  diphtheria,  there  is  not  satisfac- 
tory proof  that  they  are   necessarily   connected  with 
minute  vegetable  organisms. 

III.  That  the  real  nature  of  these  causes  is  still 
uncertain. 

It  will  now  be  advantageous,  I  think,  at  this 
point,  to  give  a  chronological  summary  of  the  events 
that  have  succeeded  one  another  in  the  history  of 
bacteriology,  from  1876  to  date.  About  the  time  that 
the  foregoing  paper  was  written,  Pasteur  had  busied 


—  9  — 

himself  with  the  serial  cultivation  of  bacteria,  or 
microbes  as  he  now  called  them.  His  object  was  to 
isolate  each  variety  completely,  and  after  a  series  of 
cultivations  in  different  media,  prove  the  actuality  of 
his  views  by  inoculation  upon  animals.  Robert  Koch, 
of  Berlin,  followed  closely  in  the  wake  of  Pasteur,  and 
devoted  himself  to  anthrax;  while  Pasteur  took  up  an 
entirely  new  topic,  viz.,  chicken  cholera.  But  his  ob- 
ject was  not  merely  to  find  the  real  cause  of  the  dis- 
ease; he  also  hoped  to  secure  what  he  termed  a 
vaccine,  borrowing  a  name  from  the  modified  virus  of 
small-pox,  or  cow-pox.  About  the  same  time  Koch* 
also  devoted  himself  to  experiments  in  septicaemia 
and  pyaemia,  by  inoculating  animals  with  putrefying 
vegetable  matter. 

In  1879  Neisserf  found  in  the  discharges  of  gonor- 
rhoea a  peculiar  microphyte  that  he  named  the  gonococ- 
cus.  In  1880  EberthJ  proclaimed  anew  the  bacterial 
nature  of  the  typhoid  fever  poison.  In  1882  Koch§  de- 
scribed his  tubercle  bacillus  and  offered  the  most  satis- 
factory proof  of  the  bacterial  origin  of  pulmonary 


*  Koch,  Ueber  d.  Aetiol.  d.  Wund-infections  krankheiten, 
Leipsig,  1878. 

f  Neisser.  Ueber  den  Pilz  der  Gonorrhoea,  Med.  Cen- 
tralbl.,  1879,  No.  28. 

\  Eberth,  Die  organismen,  etc.,  beim  Typhus  Abdom., 
Virchow's  Archiv.,  Bd.  81,  1880,  p.  58. 

§  Koch,  Die  Aetiol.  der  Tuberculose,  Ber.  Klin.  Woch., 
231,  1882. 


10    

tubercle  that  had  been  made.  In  1883  Fehleisen* 
found  a  bacillus  in  erysipelas,  which  he  regarded  as 
pathogenetic,  and  in  the  same  year  Babesf  described  a 
special  bacillus  in  lepra. 

The  year  1883  was  remarkably  prolific  of  bacte- 
riological research,  for  it  was  then  that  FriedlaenderJ; 
called  attention  to  the  coccus  of  lobar  pneumonia,  and 
Pasteur  and  Thuillier  following  Klein  substantiated 
his  views  as  to  the  bacterial  character  of  the  virus 
in  hog-cholera.  In  1884  Laveran§  published  his 
studies  on  the  microzymes  of  malarial  fever,  and  in 
1885  Lustgarten  I  gave  a  special  method,  by  which  he 
claimed  that  the  syphilitic  poison  could  be  shown  to 
have  a  special  parasite. 

It  is  generally  admitted  by  bacteriologists  that 
much  of  the  work  just  alluded  to  will  require  revision, 
a  statement  that  naturally  is  applicable  to  almost  all 
new  work  on  any  special  topic,  but  it  is  plain  that  the 
evidence  is  not  so  strong  in  any  case,  as  to  the  bacte- 
rial origin  of  the  disease  in  question,  as  in  the  case  of 
anthrax,  tubercle  alone  accepted.  Meanwhile  it  is 
proper  to  note  that  this  bacteriological  work  has,  in  a 
measure,  been  parallelled  by  physico-chemical  re- 
searches which  have  grown  out  of  these  studies,  or 


*  Fehleisen,    Die    Etiologie   d.   Erysipelas,    Berlin,  1883. 

f  Babes,  Arch,  de  Phys.,  1883. 

\  Friedlaender,  Fortschritte  d.  Med.,  Bd.  i,  1883. 

§  Laveran,  Traite  d.  fievres  palusters,  1884. 

|  Lustgarten,  Syphilis-bacillus,  1885,  Wien. 


—  II  — 

have  been  made  independently,  the  tendency  of  which 
is  to  turn  our  attention  back  towards  the  physico- 
chemical  theory,  of  which  a  passing  notice  has  already 
been  made  in  my  introductory  statements.  Some  years 
ago,  Gautier,  of  France,  and  Selmi,  of  Bologne,  appear 
to  have  made  a  simultaneous  discovery  of  certain 
chemical  substances  that  are  capable  of  infecting  the 
system  by  a  peculiar  poison.  Selmi  found  these  sub- 
stances in  the  scrapings  of  skeletons.  These  matters 
possessed  definite  crystallizable  and  physiological  prop- 
erties. Some  of  these  could  be  extracted  by  ether, 
and  some  by  chloroform.  They  were  organic  sub- 
stances, producing  with  sulphuric  acid  a  violet-red 
color,  and  giving  out  an  odor  like  hawthorn,  when 
heated.  More  recently  some  excellent  studies  have 
been  carried  out  on  the  subject  of  hog-cholera,  in  the 
Bureau  of  Animal  Industry,  in  Washington,  by  Drs. 
Salmon  and  Theobold  Smith,*  and  their  conclusions 
are  that  the  poison  of  the  disease  in  question  is  not 
merely  found  in  the  special  microphyte  of  the  disease, 
but  in  a  peculiar  substance  that  is  elaborated  by  them. 
The  conclusions  are  as  follows: 

1.  Immunity  is  the  result  of  the  exposure  of  the 
bioplasm  of  the  animal  body  to  the  chemical  products 
of  the  growth  of  the   specific  microbes,  which  consti- 
tute the  virus  of  contagious  fevers. 

2.  These  particular  chemical  products  are  pro- 

*  Proceedings   of   the  Biolog.   Soc.  of  Wash.,   Vol.  Ill, 
1884-6. 


12    

duced  by  the  growth  of  the  microbes  in  suitable  cul- 
ture liquids  in  the  laboratory,  as  well  as  in  the  liquids 
and  tissues  of  the  body. 

3.  Immunity  may  be  produced  by  introducing 
into  the  animal  body  such  chemical  products  as  have 
been  produced  in  the  laboratory. 

At  this  point  I  think  it  well  to  state  that  I  fully 
agree  with  Cornil  '£  Babes,  who  have  written  the  most 
elaborate  and  perhaps  the  most  satisfactory  work  on 
the  subject,  when  they  recommend  the  reader  to  be 
cautious  in  his  acceptance  of  much  that  has  been 
written  on  bacteriology,  and  it  is  for  the  following 
reasons:  The  morphology  of  bacteria  has  not  been 
completed,  in  a  botanical  sense;  the  territory  in  which 
they  live  and  move  is  an  uncertain  one;  animal  experi- 
mentation is  a  delicate  matter,  and  offers  large  oppor- 
tunities for  controversy;  the  technique  of  this  kind  of 
work  is  peculiarity  difficult,  even  for  one  who  is  versed 
in  ordinary  laboratory  methods,  and  finally,  the  pro- 
blems relating  to  the  chemical  nature  and  properties 
of  the  substances  elaborated  by  bacteria  have  still  to 
be  solved. 


CHAPTER  II. 

THE  MICROSCOPE  AND  MICROSCOPICAL  APPLI- 

ANCES FOR  USE  IN  A  BACTERIOLO- 

GICAL LABORATORY. 

The  Microscope.  —  It  is  necessary  to  have  an  un- 
usually good  instrument  for  the  study  of  bacteria,  and, 
indeed,  systematic  work  of  this  kind  requires  the  use 
of  immersion  lenses  of  good  quality,  together  with  an 
achromatic  condenser  of  approved  pattern.  At  the 
present  time,  Zeiss,  of  Germany,  stands  pre-eminent  as 
a  manufacturer  of  these  lenses,  and  there  is  reason  to 
believe  that  he  will  carry  their  construction  to  a  still 
higher  degree  of  perfection.  Meanwhile,  other  Ger- 
man opticians,  and  some  in  England  and  America* 
make  satisfactory  glasses.  Those  that  meet  with  most 
favor  are  the  ^,  T^,  and  ^,  but  it  should  always  be 
remembered  that  novices  in  microscopical  studies  can- 
not work  advantageously  with  such  high  powers,  and 
that  considerable  practice  with  lower  powers  and  the 
general  technique  of  the  microscope  should  precede 
all  attempts  to  achieve  success  in  the  higher  field  of 
practical  bacteriology. 

The  best  model  of  an  achromatic  condenser  has 
been  furnished  by  Abbe",  but  most  microscope  makers 
make  condensers  that  are  sufficiently  good  for  most 
work  of  the  kind. 


*  J.  W.  Grunow,  Optician,  of  70  W.  agth  St.,  New  York 
City,  makes  a  good  immersion,  -fa,  for  $60. 


—    14  - 


Fig.    i. — A    Grunow    Microscope    for    Bacteriological   Study, 

Fitted  with  Immersion  Lenses,  Nose-piece,  and 

an  Abbe  Condenser. 


Quite  recently  Zeiss  has  introduced  novelties  in 
his  apochromatic  objectives  and  compensating  eye -pieces 
for  this  especial  work.  In  the  construction  of  his 
lenses  a  new  kind  of  glass  has  been  made  under  the 
supervision  of  Prof.  Abbe.  Each  apochromatic  ob- 
jective is  to  be  used  with  a  compensating  eye-piece, 
which  both  corrects  the  former  and  gives  it  greater 
working  distance.  The  chromatic  aberration  is  said 
to  be  totally  abolished,  and  the  spherical  aberration 
materially  modified,  while  the  natural  colors  of  the  ob- 
ject examined  are  preserved.  These  objectives  are 
usually  made  to  order,  and  are  only  adapted  for  a 
particular  length  of  draw-tube.  The  highest  powers 
as  yet  in  the  market  are  the  \  and  -fa  immersion. 
Seven  varieties  of  eye-pieces  are  made,  with  powers  of 
magnification  from  i  to  27. 

From  personal  examination  I  can  recommend 
these  glasses  most  highly.  The  term  apo-chromatic 
means  merely  that  the  objective  is  so  corrected  as  to 
remove  all  color,  and  the  term  compensation  as  re- 
ferring to  an  eye-piece  means  that  the  aberrations, 
especially  the  spherical,  are  remedied  by  the  eye- 
piece rather  than  by  the  lens.  As  a  result,  he  is  able 
to  use  a  wide-angled  anterior  lens,  and  obtains  more 
working  distance  than  usual. 

Microscopic  Appliances  and  Accessories  — The  fol- 
lowing list  embraces  the  most  important  appliances 
for  a  laboratory: 

i.  Glass  bottles  with  ground  stoppers  for  the 
alcoholic  solutions  of  aniline  dyes. 


i.  Glass  bottles  with  glass  funnels  for  the 
watery  solutions  of  aniline  dyes. 

3.  A   number  of  watch-glasses  of  rather  large 
size  for  staining   sections,  or   gallipots   with   flat  bot- 
toms. 

4.  Glass  slides  and  glass  covers  of  the  best  qual- 
ity, and  as  thin  as  can  conveniently  be  used. 

5.  Holders  for  platinum  needles  and  the  ordin- 
ary milliner's  needles. 

6.  One  or  more  section  lifters,  of   which  there 
are  many  patterns. 

7.  Finely-pointed  forceps,  known  as  the  iris  for- 
ceps. 

8.  Collapsable  boxes  for  Canada  balsam. 

9.  Boxes    for    preparations.      (All  microscope- 
makers  can  furnish  these  in  great  variety.) 

10.  Labels  for  slides. 

11.  The  immersion  lens  should  be  provided  with 
a  glass-stopped  vial  for  the  special  immersion   fluid 
that  is  used.     Zeiss  uses  cedar  oil. 

The  Microtome. — This  instrument  is  now  used  in 
all  laboratories,  as  it  institutes  great  precision  in  the 
cutting  of  sections,  and  obviates  waste  of  material. 
There  are  many  kinds  of  microtome  made,  both 
abroad  and  at  home.  Unfortunately,  they  are  still 
quite  expensive.  The  instrument  known  as  Schanz's* 


*  These  microtomes  may  be  purchased  from  Meyrowitz 
Brothers,  corner  of  23d  St.  and  4th  Ave.,  N.  Y.  City. 


—  i8  — 

is  very  highly  esteemed,  and  so  also  is  Thoma's*,  but 
any  one  of  the  many  can  be  made  to  operate  suc- 
cessfully after  the  student  has  acquired  a  certain 
amount  of  familiarity  with  its  use.  It  is  best  to  have 
a  microtome  that  has  a  freezing  attachment,  so  that 
cuts  may  be  made  from  fresh  material. 

Reagents. — The  following  are  desirable: 

1.  Ordinary  alcohol  of  90  per  cent,  strength. 

2.  Absolute  alcohol. 

3.  Oil  of  cloves  or  of  bergamot  for  clarifying 
sections. 

4.  Celloidine  to  be  used  for  mounting  material 
for  cutting  in  the  microtome.     Celloidine  dissolves  in 
equal  parts  of  ether  and  alcohol,  and  should  be  kept 
in  a  wide-mouthed  bottle. 

5.  Gelatine  of  good  quality. 

6.  Acetic  acid. 

7.  Aqua  ammonia.- 

8.  Glycerine. 

9.  Nitric  acid. 

10.     Distilled  water. 


*  The  principal  advantage  of  the  Thoma  microtome  is  the 
uniformity  of  the  sections  which  can  be  made  with  it.  Sections 
of  large  size,  up  to  three  or  four  inches  in  diameter,  which  can 
t>e  cut  with  the  large  instruments,  will  be  found,  as  a  rule,  to 
te  of  a  remarkably  even  thinness,  and  they  are  cut  with  such 
•mechanical  precision  that  comparatively  few  have  to  be  dis- 
carded. Hence,  a  large  number  of  excellent  sections  can  be 
cut  from  a  single  specimen  in  a  short  time. 


—  19  — 

11.  Sterilized  water. 

12.  Ashphalt  lac. 

13.  Canada  balsam  dissolved  in  zylol. 

14.  Acetate  of  potash,  concentrated  solutions, 
and  the  following: 

Glycerine  gum  (Farrant's  solution): 

Glycerine 

Water 

Saturated  solution  of  arsenious  acid 

Take  of  these  equal  parts;  mix  and  add  solution  of  gum 
arabic  a  half  part. 

Glycerine-gelatine  (Klebs').     This  is  made  in  the 
following  way: 

Take  of  washed  gelatine 10  parts. 

Having  added  distilled  water  to  the  gelatine,  pour  off  the 
excess  of  water,  melt  the  gelatine,  and  then  add: 

Glycerine 10  parts. 

Finally,   add  a  few    drops   of  carbolic   acid   to   prevent 
moulding. 

Acidulated  alcohol: 

Alcohol 10  parts. 

Hydrochloric  acid I  part. 

Aniline  water: 

Distilled  water 100  parts. 

Aniline 5       " 

Muller's  fluid: 

Bichromate  of  potash 2  parts. 

Sulphate  of  sodium i  part. 

Distilled  water 100  parts. 


2O    

Osmic  acid: 

Distilled  water 100  parts. 

Osmic  acid 5 

Osmic  acid  should  be  placed  in  a  stoppered  black  bottle 

and  protected  from  the  light,  and  be  put  in  a  cool  place. 

Iodine  solution: 

Iodine i  part. 

Iodide  of  potassium 2  parts. 

Distilled  water 5° 

Alum-carmine: 

Carmine i  part. 

Five-per  cent,  solution  of  alum  . . .  100  parts. 

Picro-carmine  (Ranvier): 

Carmine i  part. 

Distilled  water 10  parts. 

Solution  of  ammonia 3 

Magenta  solution  (Gibbes'): 

Magenta 2  parts. 

Aniline  oil 3 

Alcohol  (Sp.  Gr.  .830) 20 

Distilled  water 20 

Gentian  violet: 
(a)    Saturated  alcoholic  solution 


(b)    Aqueous  solution 

Gentian  violet 2.25  parts. 

Distilled  water 100 

Hsematoxylon  solution. 


—     21    

Eosine: 

(a)    Saturated  alcoholic  solution 

(6)    Aqueous  solution 

Distilled  water 100  parts. 

Eosine 5       " 

Bismarck  brown: 

(a)    Concentrated  solution  in  equal  parts  of  glycerine  and 
water,  or  in  water  only. 

(&)    Aqueous  solution 

Bismarck  brown 2  parts. 

Alcohol 15       " 

Distilled  water 85 

Ebner's  solution.     This  consists  of  the  following 
ingredients: 

Hydrochloric  acid 5  parts. 

Alcohol 100      " 

Distilled  water 20       " 

Chloride  of  sodium 5       " 

Neelsen's  solution: 

Dissolve  fuchsine i  part. 

In  a  5-per-cent.  watery  solution  of 

carbolic  acid 100  parts. 

Add  alcohol 10       " 

Gibbes'  solution: 

Rosaniline  hydrochlorate 2  parts. 

Methylene  blue I  part. 

Triturate  in  a  glass  mortar. 

Dissolve  aniline  oil 3  parts. 

In  rectified  spirit 15       " 

Add  slowly  to  the  above. 

Slowly  add  distilled  water 15       " 

Then  keep  in  stoppered  bottle. 


—    22    

Fuchsine: 

(a)    Saturated  alcoholic  solution. 

(£)    Aqueo-alcoholic  solution. 

Fuchsine 2  parts. 

Alcohol 15 

Water 85       " 

Methylene  blue: 

(a)  Concentrated  alcoholic  solution. 

(b)  Aqueo-alcoholic  solution. 

Methylene  blue 2  parts. 

Alcohol 15       " 

Water 85 

Koch's  solution: 

Con.  alcoholic  solution  of  methy- 

lene  blue i  part. 

Ten-per-cent.  potash  solution 2  parts. 

Distilled  water 200  ' ' 

Loeffler's  solution: 

Con.  alcoholic  solution  of  methe- 

lene  blue 30  parts. 

Solution  of  potash,  1-10,000 100       " 

Methyl  violet: 

(a)  Con.  alcoholic  solution. 

(b)  Aqueous  solution. 

Methyl  violet 2.25  parts. 

Distilled  water 100  " 

(c)  Koch's  solution. 

Aniline  water 100  parts. 

Alcoholic  sol.  of  meth.  viol n       " 

Absolute  alcohol 10       " 


—  23  — 
Picric  acid: 

(a)    Con.  alcoholic  solution. 
(£)    Saturated  aqueous  solution. 

Kleinenberg's  solution: 

Saturated  watery  solution  of  picric 

acid 100  parts. 

Strong  sulphuric  acid 2       " 

Filter  and  add: 

Distilled  water 300       " 

Orseille  (Wedl): 

Dissolve  pure  ammonia  free  orse- 

ille  in  absolute  alcohol 20  parts. 

Acetic  acid 5       " 

Distilled  water 40       " 

Until  a  dark  red  liquid  results,  then  filter. 

Safranine: 

(a)  Concent,  alcoholic  solution. 

(b)  Watery  solution,  i-per-cent. 

Vesuvine: 

(a)  Concent,  alcoholic  solution. 

(b)  Watery  solution. 

All  the  material,  instruments,  microscopes,  etc., 
needed  for  bacteriological  study,  can  be  obtained  from 
G.  Koenig,  Berlin,  N.  W.  35  Dorotheen  Strasse,  Ger- 
many. Chemicals,  nutrient  gelatine,  etc.,  can  be  ob- 
tained from  Dr.  Georg  Gruebler,  Leipsig,  17  Dufour 
Strasse.  He  also  furnishes  Grenacher's  borax-carmine, 
Weigert's  picro-carmine,  and  Orth's  picro-lithium-car- 
mine.  Many  of  these  special  reagents  and  appliances 
can  be  obtained  from  Meyrowitz  Bros.,  23d  St.  and 
4th  Ave.,  New  York  City. 


SPECIAL  APPLIANCES  FOR  STERILIZING.* 

STERILIZING    APPARATUS. 

The  Steam  Sterilizer  (Fig.  3). — This  is  a  cylindri- 
cal vessel  of  tin,  covered  with  a  jacket  of  thick  felt.  A 
lid  or  cap  similarly  covered  fits  over  the  top.  From 
its  summit  projects  a  thermometer. 
The  interior  of  the  vessel  is  pro- 
vided with  a  wire  grating,  or  a 
series  of  holes,  to  support  the  ma- 
terial to  be  sterilized.  After  the 
chamber  has  been  fillled  to  the 
desired  height  with  water,  heat 
should  be  applied  by  one  or  more 
gas  burners.  The  water  should 
only  reach  to  the  point  indicated 
upon  the  water-gauge.  When  the 
thermometer  registers  a  tempera- 
ture of  100°  C.,  then  the  material 
to  be  sterilized  should  be  intro- 
duced, but  not  before.  The  steril- 
izer is  employed  for  sterilizing 
nutrient  media,  whether  in  boxes 

or  flasks,  for  cooking  potatoes,  or 
Fig   3      Koch's  Steam  .  ' 

Sterilizer  var.ous  other  purposes,  as  will 

be  seen. 

*  All  of  the  apparatus  described  in  this  section  can  be 
obtained  from  Messrs.  Eimer  &  Amend,  205-211  Third  Av., 
New  York  City.  This  firm  is  now  prepared  to  furnish  the  full 
equipment  for  a  bacteriological  laboratory  and  manufacture 
any  new  apparatus  according  to  the  most  recent  models. 


—  25  — 

The  Double-walled  Hot-air  Steri'izer. — This  is  a 
square  tin  box  with  double  walls,  and  designed  either 
to  stand  upon  four  legs,  or  to  be  suspended  from  the 
wall  of  the  laboratory.  (Fig.  4.)  It  is  heated  in 
the  ordinary  way  by  a  Bunsen's  compound  or  rose 


Fig.  4.     Double-walled  Hot  air  Sterilizer. 

burner,  and  the  internal  temperature  is  indicated  by  a 
thermometer  introduced  through  an  opening  in  the 
top.  In  a  second  opening  a  gas- regulator  can  be 
placed.  The  apparatus  is  used  for  sterilizing  at 


—    26    — 


higher  temperatures  than  are  practicable  by  the  steam 
sterilizing  method  first  described.  It  is  specially 
adapted  for  sterilizing  the  vessels  designed  for  re- 
ceiving the  material  used  for  purposes  of  study,  such 
as  the  receivers  for  blood  serum,  glass  flasks,  glass 
plates  for  nutrient  media,  accessory  appliances,  etc. 
In  using  this  sterilizer,  close  the  sterilization  tubes 
with  a  plug  of  clean  cotton,  and  then  place  them  in 
the  galvanized  frame  (Fig.  5),  place  the  frame  in 
the  sterilizer  and  heat  for  one 
hour  to  140°  C.  It  will  be 
found  that  this  degree  of  heat 
will  slightly  char  the  cotton 
plug.  When  glass  plates  are 
used  for  the  study  of  water,  as 
will  be  seen  at  another  place, 
they  are  to  be  put  in  a  covered 
iron  box  (Fig.  6),  and  heat- 
ed, as  before,  to  140°  C.  for 
one  hour.  Erlenmeyer's  flasks 
which  are  large  glass  flasks 
used  as  storage  vessles  for 
nutrient  media,  whether  containing  gelatine  or  agar- 
agar,  are  not  placed  in  the  galvanized  basket,  but  di- 
rectly in  the  sterilizer. 

The    Water  Bath.—  This   is  the  ordinary  water 
bath  that  is  used  in  chemical  laboratories. 

The  Hot-water  Filter. — This  is  a  heavy  copper 
funnel  having  do'uble  walls,  the  space  between  them 


The  basket- rack  for  re- 
ceiving the  steriliza- 
tion tubes. 


—  27  — 

being  filled  with  hot  water.  A  glass  funnel  is  placed 
in  the  copper  funnel,  and  its  stem,  which  is  made  to 
project  beyond  the  stem  of  the  copper  funnel,  is  held 


Fig.  6.  The  metal  box  for  glass  plates  that  are  to  be 
sterilized. 

by  a  rubber  plug  that  fits  in  the  copper  stem.  The 
water  in  the  copper  funnel  is  heated  by  a  gas  flame 
applied  to  a  lateral  projection  of  the  funnel.  It  stands 
upon  three  legs.  (Fig.  7.) 

There  is  another  and  more  recent  model  of  hot- 
water  filter,  in  which  the  copper  and  glass  stems  are 
longer  than  in  the  filter  just  described,  the  heat  being 
applied  to  the  funnel  by  a  circular  burner,  which  at 
the  same  time  serves  as  a  funnel-ring.  There  should 
also  be  on  hand  a  goodly  supply  of  glass  funnels  of 
various  sizes,  test-tubes  and  flasks,  cylindrical  gradu- 
ates marked  off  in  cubic  centimeters,  aud  a  good  bal- 
ance with  gram  weights.  The  gelatine  for  use  should 
be  of  the  best  quality,  and  the  "  gold  label  "  is  gen- 
erally preferred.  As  gelatine  is  not  suitable  for  use 


—    28    — 

when  the  temperature  of  the  atmosphere  is  high, 
agar-agar,  or  Japanese  isinglass  is  used.  It  consists 
of  the  filaments  of  certain  algae.  There  should  also 
be  on  hand:  Table  salt,  litmus  papers,  filter  paper  of 
various  descriptions,  flannel  for  filtration  purposes, 


Fig.  7.     Hot-water    Filter. 

lactic  acid,  and  carbonate  of  soda  in  saturated  solution. 
There  will  also  be  use  for  anatomical  jars  with  wide 
mouths,  and  test-tube  stands.  Rubber  caps  for  covering 
the  cotton  plugs  are  sometimes  needed.  The  platinum 
needle  that  is  used  for  inoculation  purposes  consists 


—  29  — 

of  a  platinum  wire  several  inches  in  length,  embedded 
in  a  glass  rod.  Some  needles  are  straight,  others  are 
curved  or  looped.  The  latter  is  called  in  Germany  an 
oese.  (Fig.  8.) 


'    Fig.  8. — Platinum  Needles. 

It  is  necessary  to  have  a  number  of  shallow  glass 
covers  (Fig.  9)  and  dishes  to  serve  as  damp  chambers  in 
which  the  glass  plates  remain  while  the  process  of 
cultivation  proceeds  in  the  nutrient  media  poured 
upon  them.  The  glass  plates  are  usually  4x6  inches 
in  superficial  measurement. 

Glass  benches  are  shelves  made  of  glass  to  support 
glass  plates  or  slides  in  tiers,  when  they  are  placed  in 
the  glass  chambers.  They  are  easily  made  by  cement- 
ing glass  plates  and  slips  together,  (Fig.  10  &  u)  but 
the  best  are  made  of  but  a  single  piece  of  glass. 

Glass  rods  should  be  on  hand  in  considerable 
quantity.  They  are  used  for  spreading  the  liquified 
agar-agar  or  gelatine  on  the  glass  plates. 


-  3o  - 

APPARATUS  FOR  PREPARING  POTATOE  CULTIVATIONS. 

In  order  to  sterilize  the  instruments  used  in  pre- 
paring potatoes  for  cultivation,  it  is  customary  to  have 
a  small  iron  case  of  simple  construction,  to  contain 


Fig.  9. — Glass  dishes  with  glass  covers  forming  a  moist 
chamber,  in  which  a  glass  plate  covered  with  nutrient  gelatine 
lies  upon  a  single  bench.  The  dark  areas  in  the  gelatine  cor- 
respond with  different  cultures  of  bacteria. 

a  few  scalpels  and  other  knives  for  cutting  potatoes. 
The  box  is  then  sterilized,  together '  with  its  contents, 
by  exposure  to  dry  heat  in  the  hot-air  sterilizer  (Fig. 


Fig.  10.  Glass  Benches.  Fig.  n. 

3)    for  one  hour,  at   140°-! 50°  C.      The  instruments 
for  such  a  case  need  no  special  description. 


The  damp  chambers  in  which  the  potatoes  are 
cultivated  is  the  same  in  design  as  the  ordinary  damp 
chamber  used  for  plate  cultivations  (see  Fig.  9), 
though  they  need  not  be  so  large. 

APPARATUS  FOR  THE   PREPARATION  OF  SOLID  STERILE 
SERUM. 

First  of  all,  it  is  necessary  to  provide  one's  self 
with  a  glass  jar,  Fig.  12,  with  wide  mouth  and  glass 


Fig.  12.— Glass  Jar  for  Fig.  13. — Rack  for  hold- 

holding  Blood.  ing  Pipettes. 


—  32  — 

stopper,  for  securing  the  blood.  Then  there  should 
be  pipettes  (see  Fig.  13),  graduated  or  not,  as  the  case 
may  be,  for  transferring  the  serum  from  the  jar  to 
sterilized  vessels. 

Koch's   Steam    Sterilizing   Apparatus   for   Blood 
Serum    (Fig.   14)    consists   of    a    cylindrical    vessel, 


Fig.  14.     Koch's  Steam  Sterilizing  Apparatus  for  Blood 
Serum. 

double-walled,  covered  with  felt  and  fitted  with  stop- 
cock or  steam  guage.  The  space  between  the  walls  is 
designed  to  hold  water,  and  a  felt-covered  lid  pro- 


33 

vided  with  a  tubular  prolongation  is  connected  with 
the  water  chamber.  The  water  in  the  border  of  the 
vessel  is  heated  by  a  gas  flame  from  below.  The 
water  in  the  lid  is  heated  by  a  flame  applied  to  the 
prolongation. 

In  the  centre  of  the  cylinder  is  a  column  which 
communicates  with  the  water  chamber  of  the  cylinder, 
and  from  it  pass  four  partitions,  which  serve  to  sup- 
port the  test-tubes. 

In  the  lid  are  three  openings,  one  of  which  com- 
municates with  the  water  chamber  in  the  lid  by  which 
the  latter  is  filled  and  into  which  a  thermometer  is 
then  fixed.  In  the  centre  an  opening  admits  a  ther- 
mometer, which  passes  into  the  central  pipe  of  the 
cylinder.  Through  a  third  opening  a  thermometer 
passes  to  the  cavity  of  the  cylinder. 

The  apparatus  5s  supported  on  legs.  The  method 
of  procedure  is  as  follows:  Fill  both  body  and  cover 
of  the  apparatus  with  water,  being  careful  to  keep  the 
inner  space  perfectly  dry.  Use  two  burners  and  raise 
the  temperature  to  60°  C.  The  blood  serum  which 
has  previously  been  placed  in  the  sterilization  flasks 
(already  sterilized  by  apparatus  No.  4)  is  now  de- 
posited in  the  hollow  space  of  the  apparatus,  and  the 
cover  is  replaced.  The  temperature  is  now  raised  to 
58°  C.  The  serum  should  remain  liquid,  and,  there- 
fore, the  temperature  is  not  to  be  raised  above  60°  C. 
In  order  to  solidify  the  serum,  a  special  apparatus  has 
been  devised  by  Koch  (Fig.  15).  It  consists  of  a 


—  34  — 

rather  shallow  tin  box,  with  glass  cover,  and  stands  on 
four  legs.  The  whole  is  jacketed  with  felt.  Like 
some  of  the  other  vessels  already  described,  it  is 
double-walled,  and  the  water  is  heated  from  below  by 
the  flame  of  a  gas  burner.  Of  the  four  legs  the  front 
ones  rest  in  grooves  of  the  box,  and  slide  up  and 
down  so  that  the  box  can  be  depressed  in  front  to  a 
limited  extent.  The  box  is  filled  with  from  50  to  100 


Fig.  15. — Apparatus  for  Solidifying  Blood  Serum. 

tubes  of  a  nutrient  media,  such  as  serum,  gelatine, 
or  agar-agar,  and  it  is  intended,  by  inclining  the  front 
of  the  box,  to  give  a  sloping  surface  to  the  nutrient 
media.  To  use  the  apparatus,  place  the  tubes  con- 
taining the  nutrient,  after  sterilization,  in  the  appa- 
ratus, inclining  each  tube  at  the  desired  angle;  then 
heat  to  65°  or  75°  C.  The  serum  should  then  become 
a  transparent  fluid,  and  so  remain,  and  the  heat  should 
be  applied  until  this  result  is  attained.  The  small 
quantity  of  water  which  forms  is  allowed  to  remain. 


—  35  — 

This  is  called  the  water  of  condensation,  and  has  an 
important  office.  Glass  capsules  are  small  cubes  of 
crystal  glass,  hollowed  out.  They  are  used  for  culti- 
vations in  solid  serum,  gelatine,  or  agar-agar.  They 
are  made  of  white  or  colored  glass,  and  are  fitted 
with  glass  covers. 

APPARATUS     FOR     STORING,     AND     FOR     CULTIVATING 
IN    LIQUID    MEDIA. 

Lister  s  Flasks. — These  are  globe-shaped  flasks 
with  two  necks— a  vertical  and  a  lateral  one.  The 
lateral  one  is  a  bent  spout  tapering  towards  the  ex- 
tremity. A  drop  of  liquid  remains  behind  in  the  end 
of  the  nozzle,  which  prevents  regurgitation  of  air 
through  the  spout  when  the  vessel  is  restored  to  the 
erect  position,  after  some  of  its  contents  have  been 
poured  out.  A  cap  of  cotton  wool  is  placed  over  the 
orifice,  and  the  residue  kept  for  further  use.  The 
vertical  neck  of  the  flask  is  plugged  with  sterilized 
cotton  wool,  in  the  ordinary  way. 

Sternbergs  Bulbs.—  These  are  bulbs  provided 
with  slender  necks  drawn  out  to  fine  points,  and  her- 
metically sealed. 

Aitkens  Test-tube. — This  has  been  advised  for 
counteracting  the  danger  of  the  entrance  of  atmos- 
pheric germs  on  removal  from  the  ordinary  test-tube  of 
the  cotton  wool  plug.  Each  test-tube  has  a  lateral  arm 
tapering  to  a  fine  point,  which  is  hermetically  sealed. 


-  36  - 

APPARATUS    FOR    INCUBATION. 

There  are  several  kinds  of  incubator;  most  of 
them  are  rectangular  chests-  with  glass  walls  front  and 
back,  or  in  front  only.  A  cylindrical  model  is  pre-' 
ferred  by  some.  The  two  described  here  are  those  of 
D'Arsonval  and  Babes.  The  former  admits  of  very 
exact  regulation  of  temperature,  while  the  latter  is  a 
very  practical  form  for  general  use. 

D'ArsonvaVs  Incubator. — The  "  Etuve  D'Arson- 
val" (Fig.  16)  is  a  very  efficient  apparatus,  and  is  pro- 
vided with  a  heat  regulator,  which  enables  the  tem- 
perature to  be  maintained  with  a  minimal  variation. 
It  consists  of  a  cylindrical  copper  vessel  with  double 
walls  enclosing  a  wide  inter-space  for  containing  a 
large  volume  of  water.  The  roof  of  the  water-chamber 
is  oblique,  so  that  the  wall  rises  higher  on  one  side 
than  on  the  other.  This  admits  of  the  inter-space 
being  completely  filled  with  water.  At  the  highest 
point  is  an  opening  fitted  with  a  perforated  rubber 
stopper,  through  which  a  glass  tube  passes.  The 
mouth  of  the  cylinder  itself  is  horizontal,  and  is  closed 
by  a  lid,  which  is  also  double-walled  to  contain  water. 
In  the  lid  are  four  openings;  one  serves  for  filling  its 
water-chamber,  and  the  others  for  thermometers  and 
for  regulating  the  air  supply  in  the  cavity  of  the 
cylinder.  The  cylinder  is  continued  below  by  a  cone, 
also  double-walled,  and  there  is  a  perforated  grating 
at  the  line  of  junction  of  the  cylinder  and  cone.  The 
cone  terminates  in  a  projecting  tube  provided  with  an 


—  37  — 

adjustable  ventilator.    The  apparatus  is  fixed  on  three 
supports,  united  to  one  another  below.     One  of  them 


Fig.   16. — D'Arsonval's  Incubator. 

is  utilized  for  adjusting  the  height  of  the  heating  ap- 
apparatus    (Fig.    16),    attached    to    a   circular-lipped 


aperture  in  the  outer  wall  of  the  incubator.  To  the 
lip  is  fixed  with  screws  the  corresponding  lip  of  a 
brass  box,  with  a  tightly  stretched  diaphragm  of  india- 
rubber  intervening.  Thus  the  diaphragm  separates 


Fig.  17. — Babes'  Incubator. 

the  cavity  of  the  box  from  the  water  in  the  inter-space 
of  the  incubator.  The  cap  of  the  box,  which  screws 
on,  is  bored  in  the  centre  for  the  screw-pipe  by  which 
the  gas  is  supplied.  Another  pipe  entering  the  box 
from  below  is  connected  with  the  gas-burners.  Aroun d 


~  39  ~ 

the  end  of  the  screw-pipe  a  collar  loosely  fits  and  is 
pressed  against  the  diaphragm  by  means  of  a  spiral 
wire  spring.  Close  to  the  mouth  of  the  screw-pipe  a 
small  opening  exists,  so  that  the  gas  supply  to  the 
burners  is  not  entirely  cut  off  even  when  the  diaphragm 
completely  occludes  the  mouth  of  the  screw-pipe. 

Babes'  Incubator. — The  pattern  of  Dr.  Babes'  is 
very  simple,  and  is  now  used  in  most  laboratories. 
(Fig-  17-) 

It  consists  of  a  double-walled  chest  with  sides 
and  roof  jacketed  with  felt.  Water 
fills  the  interspace  beween  the 
walls,  and  on  the  roof  are  two 
apertures,  one  for  a  gas  regulator 
and  the  other  for  a  thermometer. 
In  front  the  chest  is  closed  in  by 
a  sheet  of  felt,  a  glass  door,  and  a 
sliding  panel.  The  apparatus  can 
be  suspended  on  the  wall  or  sup- 
ported on  legs,  and  is  heated 
from  below  by  means  of  protected 
burners.  The  gas  should  pass 
through  a  thermo-regulator  to  the 
burners.  Reichert's  thermo-regu- 
lator (Fig.  1 8)  is  very  generally 
used  and  it  will  maintain  the  heat 
at  a  very  equable  temperature. 
It  consists  of  a  vertical  glass  tube 
(Fig.  1 8  c)  with  a  rectangular  arm 
fitted  with  a  pressure  screw  (s) 


Fig.    18. — Reicheri's 
Thermo-regulator. 


—   40  — 

and  another  rectangular  arm  (b).  The  gas  enters  by 
the  tube  (a)  which  is  inserted  in  the  tube  already  de- 
scribed. As  the  heat  expands  the  mercury  in  the 
vertical  tube  (<:),  the  column  rises  .and  diminishes  the 
volume  of  gas  entering;  as  the  column  falls  more  gas 
again  enters,  and  so  as  the  column  rises  and  falls  the 
supply  of  gas  is  increased  or  diminished. 

The  pressure-screw  (s)  also  assists  in  securing  the 
precise  amount  of  heat  that  is  necessary,  by  the  press- 
ure it  makes  upon  the  column  of  mercury. 

GENERAL     LABORATORY     REQUISITES. 

Siphon  Appa'atits. — Two  halfgallon  or  gallon  bot- 
tles with  siphons  connected  with  long  flexible  tubes  pro- 
vided with  glass  nozzles  and  pinchcocks  should  be 
employed  for  the  following  purposes:  One  is  used  to 
contain  distilled  water,  with  the  nozzle  hanging  down 
conveniently  within  reach  of  the  working  table;  the 
other  should  contain  a  solution  of  corrosive  sublimate 
(i  in  1000),  and  may  be  placed  so  that  the  nozzle  hangs 
close  to  the  sink  or  basin.  The  former  is  used  instead 
of  the  ordinary  wash  bottle,  and  the  latter  for  disin- 
fecting vessels  and  hands. 

Dessicator. — The  dessicator  consists  of  a  porce- 
lain pan  containing  sulphuric  acid;  it  is  covered 
over  with  a  bell-glass  receiver.  In  the  centre  of 
the  pan  is  a  column  supporting  a  circular  frame, 
which  is  covered  with  wire  gauze.  Slices  of  potatoes 
upon  which  micro-organisms  have  been  cultivated,  are 


rapidly  dried  by  the  action  of  the   sulphuric  acid  in 
confined  air. 

Besides  the  items  already  described,  there  are 
many  others  in  use,  such  as  air-pumps,  refrigerators, 
etc.  These  are  used  for  special  investigations,  and 
too  numerous  to  detail  here. 


Fig.   19. — A  Simple  Incubator  or  Brood  Oven. 

In  Fig.  18  is  seen  another  and  simple  form  of  in- 
cubator. It  is  lined  with  lead,  covered  with  glass  and 
felt,  is  fitted  with  water-guage,  and  stop-cock  and  has 
openings  for  a  thermometer  and  gas  regulator. 


*  Most  of  the  matter  in  this  section  has  been  taken  freely 
from  Crookshank. 


CLASSIFICATION    OF    BACTERIA. 

Bacteria,  called  also  schizomycetes,  or  fission 
fungi,  have  been  classified  in  various  ways.  The 
classification  of  Zopf  is  now  in  high  estimation,  but 
none  is  destined  to  live  for  any  great  length  of  time. 
Zopf  divides  them  into  four  groups,  viz:  the  coccacceae, 
bacteriaceae,  leptotricheae,  and  cladotricheae,  /.  <?.,  the 
spherical,  rod-shaped,  filamentous,  or  branching  micro- 
cymes.  Some  prefer  the  classification  of  Fluegge. 

1.  The  first  group  of  spherical  bodies  comprises 
the  streptococcus,  or  coccus  which  forms  chains,  the 
merismopedia,  or  coccus  that  forms  plates  or  lamel- 
lae; the  sarcina,  or  packet  coccus  that  forms  colonies 
in  cubes;  the  micrococcus  that  remain  in  aggregations 
of  irregular  form;  the  ascococcus  that  form  gelatinous 
pellicles. 

2.  Under  the  group    bacteriaceae  is  found  the 
genus  bacterium,  which  is  composed  of  spheres  and 
rods,   or   rods   only,  joined  to  firm  rods;   the  genus 
spirillum,    made   out   of  screw-formed   threads;     the 
bacillus;  the  vibrio,  which  is  shaped  like  a  pin,  having 
a  nodular  extremity. 

3.  The    third    group,    leptotricheae,   comprises 
four  genera.     Most  of  them  are  found   in  water.     An 
example  is  the  leptothrix  buccalis. 

4.  Of  the  last  group,  the  cladotricheae,  only  one 
example,   the    cladothrexi   Foersteri,   occurs    in    the 
human  being;  it  is  found   in  the    lachrymal    canal  of 
the  human  eye. 


CHAPTER  III. 

METHODS  OF  EXAMINATION. 

The  new  methods  for  the  detection  of  micro- 
organisms depend  mainly  for  their  success  on  im- 
proved methods  of  illuminating  and  coloring  the 
objects.  To  obtain  the  first  we  now  use  the  immersion 
objectives  with  special  condensers,  which  have  been 
described,  and  also  aniline  colors,  of  which  the  variety 
is  infinite.  These  organisms,  which  come  under  the 
general  name  of  bacteria,  maybe  present  in  the  solid  or 
fluid  tissues  of  the  body,  and  it  may  be  necessary  to 
examine  them  in  these  media;  or  we  may  have  to  study 
them  in  certain  fluids,  called  cultivation  fluids;  or 
thirdly,  they  may  have  to  be  examined  as  they  grow 
upon  certain  solid  substances,  such  as  gelatine,  pota- 
toes, blood-serum,  etc. 

Most  of  these  organisms  have  an  extraordinary 
resistance  to  various  chemical  reagents,  and  it  is  by 
this  means  often  that  we  distinguish  them  from  other 
substances  of  similar  forms.  Thus,  they  are  not  acted 
upon  by  acids  which  usually  destroy  most  inorganic 
granules;  nor  are  they  affected  by  ether,  alcohol,  or 
chloroform,  which  destroy  fatty  granules  and  even 
crystals. 

In  bacteriological  work  one  of  the  most  essential 
considerations  is  cleanliness.  All  of  the  reagents, 
instruments,  etc.,  should  be  cleansed  before  using. 


—  44  — 

This  is  especially  necessary  for  all  slides  and  cover- 
glasses,  and  it  is  even  necessary,  somtimes,  to  leave 
these  latter  in  strong  acids,  sulphuric,  nitric,  etc.,  for 
some  hours,  after  which  they  are  to  be  washed  and 
carefully  cleansed.  So  also  in  the  employment  of 
cultivating  media,  strict  attention  should  be  paid  to 
details,  or  else  the  work  will  be  unsatisfactory. 

Examination  of  Fresh  Substances. — To  examine 
liquids  containing  bacteria,  a  drop  of  the  substance, 
such  as  blood,  pus  and  the  like,  is  to  be  transferred 
with  a  sterilized  pipette,  or  loop,  to  a  slide,  and  then 
covered  with  a  clean  cover-glass.  Fresh  tissues  may 
be  teased  with  needles  in  a  sterilized  salt  solution,  and 
then  mounted  between  the  slide  and  cover-glass. 
Cultures  on  solid  media,  such  as  potatoes  and  the 
like,  can  be  examined  in  the  same  way,  a  small  portion 
being  transferred  with  a  sterilized  needle  to  a  drop  of 
sterilized  water  on  a  slide. 

Another  method  is  as  follows:  Take  a  thin 
cover-glass,  draw  it  across  the  cut  surface  of  the  tissue 
or  the  liquid,  then  taking  another  cover-glass,  squeeze 
the  two  together,  then  separating  them;  each  one  is 
found  to  be  covered  with  a  thin  layer  of  the  material 
to  be  examined.  They  are  then  dried;  afterwards 
they  are  seized  by  the  forceps  and  passed  rapidly 
through  the  flame  of  a  Bunsen  burner,  with  the  speci- 
men on  the  upper  side  of  the  glass.  If  it  be  desirable 
to  stain  them,  drop  two  or  three  minims  of  your 
staining  fluid  on  the  film;  then,  after  a  minute  or  two. 


—  45  — 

wash  off  the  surplus  stain  with  distilled  water  by 
means  of  a  wash  bottle,  place  your  cover-glass  on  a 
slide,  soak  up  the  excess  of  water  with  filter-paper, 
and  then  examine  under  the  microscope.  If  such  a 
specimen  is  to  be  preserved  permanently,  dry  and 
mount  in  Canada  balsam  in  the  usual  way. 

Baumgarteris  Method. — Place  a  drop  or  two  of 
the  fluid  to  be  examined  on  a  cover-glass  that  has 
been  thoroughly  cleansed;  press  the  fluid  into  a  thin 
layer  with  another  cover-glass,  as  has  already  been 
described.  After  the  covers  have  been  dried  and 
passed  three  times  through  the  flame  of  a  Bunsen 
burner,  immerse  the  covers  in  a  solution  of  two  drops 
of  a  33-per-cent.  solution  of  caustic  potash  to  the 
watch-glass  of  water;  then  mount  the  cover-glass  on  a 
slide  and  examine  with  a  high  power.  The  bacteria 
will  then  be  readily  seen  as  bright  shining  bodies.  To 
prepare  specimens  that  have  been  hardened  in  alcohol, 
or  fresh  specimens,  proceed  as  follows:  Place  the 
sections  first  in  absolute  alcohol  for  several  minutes, 
then  immerse  in  ether  or  alcohol,  and  then  in  a  strong 
solution  of  acetic  acid,  then  wash  in  distilled  water, 
and  afterwards  warm  in  a  2-per-cent.  solution  of 
caustic  potash.  The  potash  dissolves  the  fat  granules 
and  the  small  crystals,  and  destroys  the  tissue,  leaving 
only  the  bacterial  bodies  for  examination. 

Ehrlictis  Method. — This  is  as  follows:  Take  5 
parts  of  aniline  oil,  add  100  parts  of  distilled  water, 
then  filter  the  emulsion  carefully.  To  the  filtrate,  in  a 


-  46  - 

glass,  add  drop  by  drop  an  alcoholic  solution  of 
fuchsine,  methyl  violet,  or  gentian  violet.  Cover- 
glass  preparations  should  be  immersed  in  this  liquid 
at  least  half  an  hour;  they  are  then  to  be  washed  for 
a  few  seconds  in  dilute  nitric  acid,  i  part  to  2  of 
water,  and  then  in  distilled  water.  Everything  now, 
except  the  bacillus,  is  removed. 

Babes'  Method. — This  is  a  rapid  method  for  ex- 
amining cultures.  A  small  portion  of  the  growth 
removed  by  a  sterilized  hook  is  smeared  on  a 
cover-glass  so  as  to  form  a  thin  film;  after  it  becomes 
dry  a  few  drops  of  an  aqueous  solution  of  methyl 
violet  are  let  fall  upon  the  film;  the  excess  of  color  is 
removed  by  a  strip  of  filter  paper. 

ffis's  Method. — This  is  also  suited  for  fresh  speci- 
mens. A  slide  is  prepared  as  has  already  been  de- 
scribed, but  the  reagents  are  applied  by  a  pipette  at 
one  edge  of  the  cover-glass,  and  withdrawn  from  be- 
neath the  cover  by  means  of  a  strip  of  filter  paper 
placed  at  the  opposite  edge. 

Cover-glass  Impressions. — A  perfectly  clean  cover- 
glass  is  placed  on  a  plate  or  potato  culture  and  gent- 
ly pressed  into  it;  it  is  then  carefully  raised  by  forceps 
and  allowed  to  dry  after  being  passed  through  the 
flame  three  times.  The  growth  in  this  way  is  bodily 
transferred  to  the  cover-glass. 

Gram's  Method. — Specimens  prepared  by  this 
method  should  be  kept  in  absolute  alcohol,  from  which 
they  are  transferred  at  once  to  Weigert's  or  Ehrlich's, 


—  47  — 

gentian  violet,  or  fuchsine  and  aniline  water  solutions, 
where  they  remain  for  several  minutes,  though  tubercle 
sections  should  be  kept  at  least  twenty-four  hours, 
afterwards  washed  for  three  minutes  in  alcohol,  and 
then  in  a  solution  of  10  parts  of  iodine,  20  parts  of 
iodide  of  potassium,  and  3000  parts  of  water,  until  a 
dark  blue  violet  is  changed  to  a  dark  purple  red. 
Now  wash  in  alcohol  until  most  of  the  color  has  dis- 
appeared, then  clarify  in  oil  of  cloves.  Such  sections 
are  to  be  mounted  at  once  in  balsam,  when  the  tissues 
will  be  seen  to  have  a  faint  yellow  color,  and  the  bac- 
teria are  deep  blue  or  black.  Most  micro-organisms 
can  be  stained  by  this  method,  but  some  require  a 
different  treatment.  For  example,  the  bacillus  of 
glanders  is  best  stained  with  an  alkaline  solution  of 
methyl  blue,  which  is  prepared  (Schutz)  by  making  a 
one-tenth  part  per  thousand  watery  solution  of  caustic 
potash  and  adding  one-third  the  bulk  of  a  saturated 
solution  of  methyl  blue.  These  specimens  are  then 
treated  with  dilute  acetic  acid,  and  mounted  in  the 
regular  way.  So  also  the  typhoid  bacilli  are  said  to 
be  stained  best  by  this  method — not  so  well  by  Gram's 
method.  For  certain  organisms,  such  as  the  micro- 
coccus  of  pneumonia  in  the  sputum,  and  the  micro- 
coccus  of  gonorrhoea,  Klein  recommends  a  mixture  of 
methyl  blue  and  vesuvin. 

Friedlaender  employes  the  following  method  for 
the  micrococcus  of  pneumonia:  He  takes  the  solution 
of  fuchsine,  i  part;  distilled  water,  100  parts;  alcohol 


and  glacial  acetic  acid,  2  parts;  immerses  the  sections 
in  it,  then  washes  them  in  alcohol,  next  in  a  2-per-cent. 
solution  of  acetic  acid,  then  in  alcohol  and  oil  of 
cloves,  and  mounts  in  balsam. 

To  Stain  Tubercular  Bacilli.  —  The  tubercle 
bacillus  takes  the  following  dyes  slowly,  but  holds 
them  steadily: 

The  method  of  Ehrlich  is  recommended  by 
Koch. 

Kaatsers  Method. — After  staining  the  film  on  the 
cover-glass  with  aniline  water  and  alcohol  gentian- 
violet  solution,  he  immerses  them  from  one-half  to  one 
minute  in  a  solution  of  alcohol,  90  per  cent.,  150 
parts;  distilled  water,  30  parts;  hydrochloric  acid,  i 
part;  he  then  washes  them  thoroughly  from  one  to 
two  minutes  in  po-per-cent.  alcohol,  until  the  coloring 
matter  disappears  from  the  film;  he  then  dries  and 
adds  a  watery  solution  of  vesuvine.  After  a  couple  of 
minutes  he  again  washes  in  distilled  water,  dries, 
and  mounts  in  balsam. 

Gibbes  Method. — This  may  be  used,  especially 
where  a  double  staining  is  desirable.  He  takes  of 
rose-aniline  hydrochloride,  2  parts;  methyl  blue,  i 
part;  triturates  in  a  glass  mortar,  then  he  dissolves 
aniline  oil,  3  parts;  in  rectified  spirits,  15  parts.  He 
then  drops  the  cover-glass  with  hardened  sputum  on 
it  in  this  solution  after  it  has  been  warmed,  and  keeps 
it  there  for  four  or  five  minutes.  He  then  washes  it 
in  alcohol  until  no  more  color  comes  away,  after 
which  he  mounts  it  in  Canada  balsam. 


—  49  — 

Weigert's  Method. — For  staining  solid  tissue  con- 
taining bacteria,  place  the  sections  from  six  to  eighteen 
hours  in  a  i  per-cent.  watery  solution  in  any  of  the 
aniline  dyes.  If  it  be  desirable  to  hasten  the  process, 
place  the  sections  in  an  incubator  and  heat  to  45°  C. 
If  a  stronger  solution  be  used,  the  sections  may  be 
over-stained.  They  are  then  treated  with  carbonate 
of  potash  in  half-saturated  solution.  Next  the  sec- 
tions are  washed  with  distilled  water  and  passed 
through  6o-per-cent.  alcohol  into  absolute  alcohol, 
when  almost  decolorized,  lift  the  sections  from  the 
fluid,  dry,  and  transfer  to  oil  of  cloves;  preserve  in 
Canada  balsam. 

To  Harden  and  Decalcify  Preparations. — In  hard- 
ening small  organs,  place  on  a  piece  of  filter-paper  at 
the  bottom  of  a  glass  jar,  and  cover  with  twenty  times 
the  volume  of  absolute  alcohol;  or,  Mueller's  fluid 
may  be  used  for  a  day  or  two,  and  then  absolute  alco- 
hol. Tissues  prepared  in  this  way  are  ready  for 
cutting  in  two  or  three  days.  Teeth  or  bones  must 
be  phced  in  a  decalcifying  solution,  such  as  Kleinen- 
berg's;  when  sufficiently  softened  they  should  be 
soaked  in  water  to  wash  out  the  picric  acid,  then 
transferred  to  alcohol. 

To  Imbed  and  Cut  Specimens.—  Material  to  be  cut 
with  a  freezing  microtome  should  be  soaked  in  water 
before  being  frozen.  In  some  instances  the  hardened 
tissues  should  be  well  eoaked  first  in  gum  mucilage 
and  then  frozen.  In  other  microtomes,  the  tis- 


sues  are  imbedded  in  paraffine  or  celloidine,  and 
mounted  on  cork;  celloidine  is  more  commonly  em- 
ployed. The  tissues  to  be  imbedded  are  placed  in  a 
mixture  of  ether  or  alcohol  for  an  hour  or  more;  they 
are  then  transferred  to  a  solution  of  celloidine  in  equal 
parts  of  ether  and  alcohol,  and  left  there  for  several 
hours.  Corks  ready  for  the  clamps  of  the  microtome 
are  covered  over  with  a  solution  of  celloidine,  then  this 
is  applied  with  a  glass  rod  to  the  surface  which  is  to 
receive  the  tissue.  The  tissue  remains  in  celloidine 
solution  from  one  to  twenty-four  hours.  In  some  tis- 
sues, as  in  the  lung,  a  longer  time  is  necessary.  A  lit- 
tle of  the  solution  which  is  of  a  syrupy  consistence, 
should  be  allowed  to  fall  on  the  tissues  so  as  to  cover 
them  completely,  and  the  mounted  specimen  is  placed 
in  a  60- or  8o-per-cent.  alcohol  to  harden  the  celloidine. 
The  specimen  will  be  ready  for  cutting  next  day. 
The  celloidine  in  the  sections  disappears  during  the 
process  of  clarifying  in  clove-oil. 


CHAPTER  IV, 

CULTURE  METHODS. 

Dr.  Koch,  of  Berlin,  has,  as  I  have  already 
shown,  introduced  improvements  in  the  investigation 
of  bacteria  by  means  of  the  process  of  cultivation, 
which  Pasteur  originated.  By  it  the  special  bacteria, 
which  are  the  cause  of  any  particular  change  in  the 
tissues  or  fluids,  may  be  eliminated,  so  that  forms  which 
are  harmless  may  readily  be  separated  from  those 
that  are  poisonous.  This  process,  which  seems  diffi- 
cult, can  readily  and  successfully  be  accomplished  if 
proper  care  be  exercised.  If,  for  example,  we  wish  to 
select  the  bacteria  which  give  rise  to  a  peculiar  colora- 
tion in  a  liquid,  we  take  a  small  quantity  of  the  liquid, 
just  enough  to  cover  the  point  of  a  needle,  and  then 
mix  it  with  about  a  teaspoonful  of  gelatine,  which  be- 
comes fluid  at  35°  C.;  for  the  bacterial  organisms 
which  have  been  taken  up  by  the  needle  are  dissemin- 
ated through  the  melted  gelatine,  which  is  then  spread 
upon  the  flat  surface,  excluded  from  the  air,  and  after 
a  variable  period,  usually  from  one  to  three  or  four 
days,  we  obtain  from  each  individual  spore  a  distinct 
culture,  some  of  one  color,  some  of  another;  these  are 
pure  cultivations.  The  next  step  consists  in  taking 
from  each  of  these  fields  and  sowing  again  in  fresh 
melted  gelatine.  If  now  we  wish  to  prove  to  ourselves 
that  we  have  discovered  the  special  parasite,  we  take 


—  52  — 

this  special  liquid,  free  from  bacteria,  and  inoculate  it 
with  the  colored  patch  that  we  suspect  is  the  cause  of 
the  coloration.  If  the  same  color  be  produced  as  in 
the  original  instance,  there  is  prima  facie  evidence 
that  we  have  discovered  the  special  parasite.  This  is 
the  general  plan  under  which  Koch  proceeded  to 
eliminate  the  common  bacillus  of  cholera  and  the  or- 
ganisms that  he  claims  are  the  causes  of  pyaemia, 
pneumonia,  etc.  This  method  of  gelatine  culture  is 
simple  enough  to  be  practiced  by  any  intelligent  ob- 
server. It  has  come  to  be  held  now-a-days,  that 
the  color  or  the  naked-eye  appearance  of  the  growth 
of  a  culture  rather  than  its  morphological  character, 
determines  the  relationship  of  a  micro-organism,  for 
the  form  of  these  bodies  is  very  changeable,  each 
species  being  subject  to  alterations  in  form,  according 
to  the  menstruum  in  which  it  is  found.  To  have  a 
culture  successful,  the  medium  must  necessarily  be 
completely  sterile;  but  this  may  be  either  liquid  or 
solid,  some  micro-organisms  growing  better  in  one 
than  in  the  other.  Thus,  it  is  said  that  the  bacteria  of 
chicken  cholera  will  die  within  48  hours  if  immersed 
in  a  decoction  of  beer-yeast,  which  is  an  excellent 
nutrient  fluid  for  putrefactive  bacteria. 

Fluid  Media. — A  common  nutrient  medium  is  a 
meat  decoction  of  any  kind.  It  should  be  free  from 
oily  matter,  of  neutral  reaction,  free  from  particles  of 
any  kind,  and  sterile.  Vegetable  decoctions  "and  in- 
fusions are  also  used,  but  less  frequently. 


—  53  — 

The  method  of  sterilizing  a  culture  medium  is 
chiefly  by  subjecting  it  to  a  temperature  sufficient  to 
kill  any  form  of  bacterium.  This  may  be  accomplished 
in  several  ways. 

Pasteur's  Method  consists  in  placing  the  culture 
fluid  in  small  pear-shaped  flasks,  which  are  blown 
from  a  glass  tube  and  then  hermetically  sealed.  These 
flasks  are  then  bathed  in  a  solution  of  chloride  of 
lime  or  nitrate  of  soda,  and  kept  there  for  twelve 
hours  at  a  temperature  of  no0  to  115°  C. 

Buchners  Method. — A  kettle  27  centimetres  in 
diameter  and  45  centimetres  deep  is  used;  this  can  be 
closed  steam-tight.  Water  is  then  poured  in  until  it 
is  5 -to  8  centimetres  deep.  The  test-tubes  are  ar- 
ranged in  tiers,  and  then  suspended  one  above  the 
other  in  the  kettle;  each  tube  is  closed  with  cotton 
and  covered  with  cloth  at  the  top.  After  the  cover  of 
the  vessel  has  been  screwed  on  tight,  the  whole  appa- 
ratus is  heater!  for  one  hour  and  a  quarter,  and  then 
kept  for  one  hour  at  110°  C. 

Miquel's  Method  consists  in  sterilizing  by  the  use 
of  filters.  For  the  larger  forms  he  uses  porous  paper; 
for  the  more  minute,  clay  or  plaster  of  Paris  cups. 
He  uses  flasks,  in  the  side  of  which,  near  the  neck,  is 
a  small  ventilation  tube.  The  neck  is  also  somewhat 
narrowed  at  its  lower  extremity.  Now  the  mixture  of 
plaster-of- Paris  and  asbestos  is  poured  into  the  neck 
of  the  flask  and  allowed  to  dry  slowly.  The  flask  is 
then  heated  until  the  air  has  been  expelled  and  the 


—  54  — 

bacteria  have  been  killed.  The  ventilation  tube  is 
then  fused  together,  and  the  culture  fluid  is  poured 
upon  the  plaster  plug  in  the  flask,  through  which  it  is 
gradually  forced.  This  method  is  used  for  the  culti- 
vation fluids  that  contain  albumen,  which  would  be 
coagulated  by  most  of  the  methods  that  have  been 
described. 

Loeffler's  Method. — This  is  the  most  popular  now 
in  use.  The  apparatus  has  already  been  described. 
This  consists  of  a  cylinder  about  half  a  metre  high, 
and  20  to  25  centimeters  in  diameter;  it  has  a  copper 
bottom  and  is  protected  by  a  felt  covering.  At  the 
lower  third  of  the  interior  is  a  grate,  and  the  space 
beneath  is  filled  three-quarters  full  of  water.  The 
heat  is  applied  by  three  or  four  gas  flames;  the  lid 
of  the  cylinder  is  covered  with  felt.  A  thermometer 
passes  through  this  lid;  it  has  the  advantage  of  not 
permitting  the  temperature  to  rise  above  100°  C. 

SOLID    OR    GELATINE    CULTURE    MEDIA. 

;  Solid  Nutrient  Media. — These  are  made  by  taking 
any  of  the  fluid  media  just  described,  and  adding  to 
them  pure  gelatine  or  agar-agar,a  material  that  be- 
comes fluid  at  a  low  temperature,  viz.,  30°  C.  The 
advantage  of  solid  media  is  that  they  enable  the  germs 
to  be  distributed  widely,  so  that  each  germ  can  go  on 
developing  apart  from  its  neighbor,  and  thus  grow  so 
as  to  form  a  considerable  field  visible  to  the  naked 
eye.  These  solid  media  are  better  nutrient  material 


—  55  — 

than  the  surface  of  a  potato,  and,  indeed,  it  is  difficult 
to  separate  the  organisms  of  infective  diseases  from 
the  putrefactive  bacteria  by  growth  on  potato,  because 
the  latter  grow  so  rapidly  and  develop  so  extensively 
that  they  soon  conceal  the  former.  When,  however, 
pure  cultivations  have  been  obtained,  they  may,  in 
some  instances,  be  made  to  grow  successfully  upon 
potato.  When  a  test-tube  is  used  for  solid  cultures, 
the  tube  should  be  allowed  to  incline  at  an  angle  of 
45  degrees,  so  that  the  surface  of  the  gelatine  will  be 
larger.  The  objection  to  gelatine  cultures  is  that  they 
will  not  remain  solid  at  the  temperature  best  adapted 
for  the  growth  of  bacteria,  namely,  30°  to  37°;  accord- 
ingly, Koch  has  devised  another  method. 

Serum  Culture. — The  serum  taken  from  the  blood 
of  an  ox  or  sheep  after  the  clot  has  been  removed,  is 
placed  in  test-tubes  closed  with  cotton  and  rubber 
cloth,  so  that  they  are  water-tight.  They  are  heated 
one  hour,  daily,  for  six  days,  at  58°  C.,  by  which  the 
serum  is  usually  sterilized.  It  is  then  heated  at  65° 
C.,  until  the  cotton  is  charred  slightly.  •  The  serum 
now  appears  as  yellow-colored,  transparent,  faintly 
opalescent,  and  firm.  It  should  permit  no  bacteria  to 
develop  for  several  days.  Material  to  be  investigated 
is  now  placed  upon  this  firm  blood-serum,  and  the 
whole  is  kept  at  37°  or  38°  C.,  until  growth  takes  place 
— in  twelve  to  fourteen  days.  Hydrocele  fluid  may 
also  be  used. 

The    Culture     Vessels. — The    common    test-tube 


~  56  - 

closed  with  cotton  is  ordinarily  used,  but  in  some 
instances  other  forms  are  recommended.  Some 
operators  take  a  conical  flask  of  glass  having  a  flat 
bottom.  They  then  employ  a  glass  tube  of  a  diameter 
sufficient  to  enter  the  neck  of  the  flask,  and  somewhat 
longer  than  it.  The  tube  is  filled  three-quarters  full 
of  asbestos  wool,  upon  which  is  placed  a  pad  of 
cotton.  It  is  lowered  rapidly  in  some  cotton  bat- 
ting and  pressed  firmly  into  the  neck  of  the  flask. 
This  apparatus,  when  heated  to  200°  C.,  is  thoroughly 
sterilized. 

Salmons  Tube. — The  culture-tube  of  Salmon 
consists  of  a  vessel  like  a  test-tube  with  heavy  glass, 
about  five  inches  in  length,  and  three  quarters  of  an 
inch  in  diameter.  Over  the  top  of  this  tube  a  hollow 
cap  is  fitted,  and  the  edges  so  ground  as  to  fit  snugly 
over  the  surface  of  the  reservoir,  which  is  also  ground. 
This  cap,  about  two  and  one-half  inches  long,  con- 
tracts near  its  middle  into  a  narrow  tube,  about  three- 
eighths  of  an  inch  in  its  internal  measurement.  A 
third  tube,  for  ventilation,  is  like  an  inclined  U,  one 
limb  about  three  inches  long,  and  one  and  one-half 
inches  longer  than  the  limb  which  fits  over  the  narrow 
tube.  The  short,  free  limb  of  the  ventilation  tube 
contains  a  plug  of  wool  one  and  one-half  to  two  inches 
long.  The  limbs  of  the  ventilation-tube  are  about 
•  one  inch  apart.  The  culture  fluid  is  introduced  by 
removing  the  cap.  The  pipette  used  to  introduce  the 
fluid  containing  bacteria  is  an  ordinary  glass  tube, 


—  57   — 

about  one  and  one-quarter  inches  in  diameter,  and  two 
or  three  inches  long,  drawn  out  into  a  fine,  almost 
capillary  Kibe,  which  should  reach  the  bottom  of  the 
reservoir  when  introduced  through  the  narrow  tube  of 
the  cap;  a  cap  of  wool  occupies  the  other  end,  which 
is  closed  by  a  rubber  ball.  The  method  of  using  it  is 
as  follows: 

The  pipette  is  first  sterilized  by  super-heating 
every  portion  of  it  from  the  top  of  the  capillary  tube 
to  near  the  rubber  ball,  until  the  air  has  been  sub- 
jected to  a  temperature  of  about  150°  C.  It  should 
be  brought  to  a  dull  red  heat.  When  cool,  the  capil- 
lary portion  should  again  be  drawn  once  or  twice 
through  the  flame.  The  ventilator  of  the  culture  tube 
containing  the  bacteria  to  be  sown  is  flamed  and  re- 
moved, and  the  narrow  tube  of  the  cap  super-heated; 
the  rubber  bulb  is  slightly  compressed  and  the  pipette 
introduced,  a  few  drops  drawn  up,  the  pipette  with- 
drawn, the  cap  again  super-heated,  and  the  ventilator 
replaced.  The  cap  of  the  fresh  tube  is  now  super- 
heated before  and  after  removing  the  ventilator;  the 
pipette  introduced,  a  drop  allowed  to  draw  into  the 
the  culture  fluid,  the  pipette  removed,  the  narrow  tube 
of  the  cap  again  flamed,  and  the  ventilator  replaced. 
This  method  dispenses  with  cotton  plugs  and  is  easily 
learned.  The  tubes  do  not  break  easily. 

Sternbergs  Flisks. — These  are  easily  made  from 
glass  tubing.  They  are  little  bulbs  blown  from  a  glass 
tube,  and  have  a  long  neck  that  tapers  gradually  to  a 


-58- 

capillary  point.  Each  flask  contains  a  sufficient 
amount  of  nutrient  fluid  and  oxygen  to  insure  a  vigor- 
ous growth  of  organisms.  When  properly  sterilized, 
the  medium  remains  closed  indefinitely,  and  the  flasks 
may  be  packed  away  in  drawers  or  boxes  for  use,  or 
carried  about  from  place  to  place.  The  inoculation 
from  one  flask  to  another,  or  from  fluid,  is  easily  ac- 
complished with  perfect  security,  or  small  amounts  of 
fluid  may  at  any  time  be  withdrawn  for  microscopic 
examination  without  the  danger  of  introducing  foreign 
organisms,  or,  indeed,  these  flasks  may  be  used  as 
syringes,  the  contents  being  forced  beneath  the  skin 
of  a  living  animal  if  only  gentle  heat  be  applied  to  the 
ball,  causing  the  air  to  extend  and  forcing  the  con- 
tents through  the  neck  of  the  flask. 

To  Introduce  a  Sterilized  Culture  Fluid  into  a 
Sterilized  Culture  Vessel. — For  this  purpose  a  sharp 
canula  is  needed,  made  of  silver,  platinum,  or  glass, 
which  is  fastened  with  a  rubber  tube  into  the  vessel 
containing  the  culture  fluid.  We  previously  sterilize 
the  canula  by  holding  it  in  steam  escaping  from  the 
vessel,  or  by  heating  it  in  the  alcohol  flame.  Remove 
the  cotton  plug  from  the  vessel  and  push  the  canula 
through  the  asbestos  into  the  vessel;  now  relax  the 
clamp  on  the  rubber  tube  and  the  culture  fluid  will 
flow  over  into  the  culture  vessel.  When  it  is  not 
necessary  to  exercise  great  precaution  against  con- 
tamination, as  in  the  pigment  bacteria,  cultures  may  be 
carried  on  under  bell-glasses  by  sowing  bacteria  upon 


—  59  - 

solids,  potatoes,  turnips,  or  eggs,  or  upon  gelatine  or 
agar-agar  upon  glass  slides. 

Inoculation  Experiments. — It  is  a  matter  of  fact 
that  certain  animals  only  are  susceptible  to  certain 
bacteria.  For  example,  birds  are  not  affected  by  an- 
thrax bacteria.  Then  again,  the  mucous  surfaces  of 
the  mammals  are  not  suitable  places  for  the  introduc- 
tion of  germs,  because  they  are  already  inhabited  by 
myriads  of  bacteria.  There  are,  however,  two  plans 
for  the  inoculation  of  pure  cultures.  The  first  is  as 
follows: 

Having  prepared  the  skin  by  the  removal  of  hair, 
or  feathers,  as  the  case  may  be,  and  having  washed  it 
with  an  antiseptic  solution,  dried  it  with  absorbent 
cotton,  and  cut  it  with  a  sterilized  knife,  the  bacteria 
of  a  pure  culture  are  introduced  by  the  sterilized  flat 
wire,  covering  the  spot  with  surgeons'  antiseptic 
gauze,  all  contamination  from  the  air  is  avoided;  or, 
the  material  may  be  injected  with  a  hypodermic 
syringe  properly  sterilized. 

So  far  as  our  present  experience  goes,  it  cannot 
be  said,  however,  that  inoculation  a-;  yet  has  produced 
very  positive  results.  Certain  it  is  that  inoculation  for 
anthrax  and  pleuro-pneumonia  in  cattle  has  not  stayed 
the  progress  of  the  disease  in  countries  where  it  has 
been  tried.  Whether  it  will  be  a  success  in  the  future 
is  a  matter  that  remains  to  be  determined. 


CHAPTER  V. 

METHODS  OF  INVESTIGATING  SPECIAL 
BACTERIA. 

ANTHRAX. 

Toussaints  Method. — In  the  year  1879,  Professor 
Toussaint  announced*  that  he  could  inoculate  animals 
with  the  virus  of  anthrax,  prepared  in  a  peculiar  way, 
and  give  entire  immunity  against  subsequent  disease. 
His  plan  was  as  follows: 

First. — After  defibrinating  the  blood  of  an  animal 
suffering  from  anthrax,  he  subjected  it  to  a  tempera- 
ture of  55°  C.  for  about  ten  minutes.  This  was  then 
inoculated  upon  a  healthy  animal. 

Second. — He  added  to  the  anthrax  blood  one- 
fourth  of  one-per-cent.  of  carbolic  acid,  and  then  in- 
oculated the  carbolized  blood. 

According  to  his  statements  the  bacteria  were 
killed,  but  the  infective  material  was  not  destroyed, 
but  was  rendered  less  virulent.  This  method  was 
subsequently  proven  to  be  fallacious. 

Pasteur  s  Method. — On  the  28th  of  February, 
1 88 1,  Pasteur,  of  Paris,  announced  to  the  Academy 
that  he  had  discovered  another  method  of  successfully 
inoculating  against  anthrax.  His  method  was  simply 
to  expose  the  anthrax  poison  to  atmospheric  air  under 

*  Comptes  Rend.  Tom.,  87,  p.  1217,  1879. 


—  6i  — 

certain  conditions.  He  further  stated  that  the  anthrax 
bacteria  reached  their  highest  development  at  a  temp- 
erature between  25°  and  40°  C.;  at  either  higher  or 
lower  temperatures  they  increased  or  developed  more 
slowly,  and  ceased  to  develop  entirely  at  a  tempera- 
ture below  15°  or  above  45°  C. 

Pasteur  found,  as  he  stated,  that  the  anthrax 
bacilli  could  be  cultivated  at  a  temperature  between 
42°  and  43°  C.  in  beef  broth  fluid;  but  when,  under 
such  circumstances,  the  fluid  was  exposed  to  filtered 
atmospheric  air,  the  bacilli  lost  their  virulence,  so  that 
in  the  course  of  fourteen  days  the  culture  might  be 
inoculated  upon  sheep  without  danger,  though  where 
the  culture  had  only  been  treated  for  twelve  days, 
one-half  the  inoculated  animals  died.  In  these  cul- 
tures, if  maintained  at  a  temperature  between  42°  and 
43°  C.,  the  bacilli  or  bacteria  survived  for  forty-six 
weeks,  and  furnished  successful  inoculations  for  pro- 
tection against  anthrax;  after  this  period  had  passed, 
they  died.  If,  however,  it  were  found  that  the  sheep 
had  been  inoculated  with  the  culture  fluid  twenty-four 
days  old,  when  again  inoculated  within  twelve  days 
with  bacteria  taken  immediately  from  an  animal  sick 
with  anrhrax,  they  died.  If  this  second  inoculation 
were  made  with  the  twelve-days-old  culture  instead  of 
the  blood,  the  sheep  were  found  necessarily  protected. 
He  found  that  the  inoculations  should  not  be  repeated 
too  rapidly;  at  least  twelve  days  should  elapse  between 
them,  lest  the  effect  be  cumulative.  Pasteur  and  his 


—    62    — 

assistants,  Chamberland  and  Roux,  usually  inoculated 
animals  upon  the  inner  side  of  the  thigh,  or  upon  the 
ear,  or  injected  the  culture  underneath  the  skin. 

Cattle  are  not  so  easily  affected  as  sheep.  Pas- 
teur anounced  that  with  sheep  80  per  cent,  had  an 
immunity  lasting  over  one  year.  It  has  been  found, 
however,  that  only  certain  animals  are  susceptible  to 
the  virus  of  anthrax.  At  this  point  it  will  be  well  to 
note  the  results  of  Pasteur's  method  in  England. 

Thus,  in  1884,  Lauder  Brunton  says,  though  we 
agree  with  Koch  and  Klein  that  immunity  can  be  con- 
ferred by  inoculation,  this  is  an  interestiong  rather 
than  a  practical  subject.  He  says,  Pasteur  has  claimed 
that  his  method  gives  absolute  immunity,  and  that  it 
is  harmless,  but  there  is  no  doubt  that  when  Pasteur 
performs  his  inoculations  without  any  deaths,  he  works 
with  cultures  too  weak  to  give  any  immunity.  His 
inoculation  fluid  vaccines  have  been  found  variable. 
Thus,  sometimes  his  vaccin  premier  has  killed  a  flock 
of  sheep,  while  the  vaccin  deuxieme  has  been  inert. 
The  objection  to  this  method  is  that  immunity  can 
only  be  conferred  by  a  percentage  of  loss  from  deaths 
greater  than  would  result  if  the  flock  were  turned 
upon  a  notoriously  infected  pasture.  And  further, 
this  inoculation  favors  the  spread  of  the  disease  by  the 
formation  of  spores  when  any  of  the  bacilli  fall  on  the 
wool  of  the  animal.  The  immunity  thus  given  lasts, 
at  the  most  favorable  computation,  no  longer  than  one 
season.  That  it  may  be  possible  in  the  future  to  dis- 


-  63  r 

cover  a  method  of  obtaining  immunity  without  too 
great  a  loss  during  the  process  must  be  allowed,  but 
to  consider  it  proven  that  we  at  present  possess  such 
a  means  in  the  method  of  inoculation  described  by 
Pasteur,  can  only  lead  to  disappointment,  etc. 

Chauveaus  Method. — Prof.  Chauveau  was  one  of 
the  first  to  demonstrate  that  the  presence  of  oxygen, 
which  Pasteur  alleged  attenuated  the  virus,  was  really 
inert  in  this  respect.  He  further  showed  that  bacteria 
of  anthrax  would  survive  a  temperature  of  47°  C., 
after  exposure  to  it  for  two  to  four  hours  at  least; 
they  were  rendered  less  harmful,  but  they  were  still 
virulent.  His  improved  method  was  as  follows: 

He  first  took  a  drop  of  fresh  infected  blood  from 
an  animal  suffering  from  anthrax,  and  then  placed  it 
in  the  culture  flask  which  contained  20  grains  of 
sterilized  broth;  there  he  retained  it  for  two  hours  at 
43°  C.;  it  was  then  heated  for  three  hours  to  47°  or 
49°  C.  He  further  showed  that  flasks  containing  one 
or  two  litres  could  furnish  sufficient  culture  virus  to 
inoculate  at  least  8,000  sheep.  He  took  one  of  these 
flasks  with  three  openings,  and  filled  it  five-sixths  full 
of  sterilized  broth.  The  middle  opening  was  armed 
with  a  long  tube  which,  descended  to  the  bottom  of 
the  flask;  this  tube,  the  outer  edge  of  which  was  filled 
with  cotton,  was  used  for  the  entrance  of  air.  Of  the 
two  side  openings,  one  is  used  as  an  adductor  tube, 
and  the  other  is  a  drawn  out  cylinder  for  emptying 
the  small  flasks.  By  this  last  tube  we  introduce  the 


-  64  — 

bacteria,  in  the  proportion  of  one  drop  to  ten  grammes 
of  culture  fluid.  After  the  introduction  of  the  germ 
fluid  the  tube  is  closed  and  passed  through  the  flame 
of  the  Bunsen  burner.  This  last  culture  fluid  is  then 
placed  in  a  thermostat  at  35°  to  37°  C.  The  develop- 
ment is  incomplete  if  the  fluid  be  at  rest,  but  when  air 
is  passed  through  it  by  means  of  the  tube,  abundant 
development  takes  place.  In  one  week  the  germina- 
tion is  complete,  and  there  is  a  rich  development  of 
spores,  whose  virulence  the  heat  has  attenuated.  The 
best  culture  fluid  is  chicken  broth,  one  part  meat  to 
four  of  water.  The  current  of  air  should  be  very  reg- 
ular. The  flask  should  be  shaken  night  and  morning. 
The  nearer  the  temperature  to  40°  C.,  the  better  the 
culture.  Third,  from  this  large  flask  the  little  tubes 
used  by  Pasteur  are  filled.  Some  are  placed  in  a 
water-bath,  others  in  an  air-bath  and  heated  to  85  u  or 
90°  C.  In  order  to  get  the  first  inoculation  (premier 
vaccin),  heat  them  to  the  highest  point  possible  short 
of  preventing  proliferation.  For  the  second  inocula- 
tion (deuxieme  vaccin),  heat  to  a  point  two  degrees 
less;  usually  84°  C.  answers  for  the  first,  and  82°  for 
the  second. 

Chamberland  and  Rouxs  Method. — Objections 
having  been  made  to  Chauveau's  method  that  his  at- 
tenuated virus  does  not  retain  its  attenuation,  Cham- 
berland and  Roux  undertook  to  improve  upon  Tous- 
saint's  method.  They  took  the  beef-broth  culture  of 
the  bacillus  anthracis,  after  it  had  been  neutralized 


with  potash,  etc.,  and  antiseptics,  they  placed  the  cul- 
ture in  an  oven  at  35°  C.  The  growth  of  bacteria 
was  greatest  where  the  least  antiseptic  had  been  used, 
and  diminished  as  the  percentage  of  the  antiseptic 
increased.  The  attenuated  bacteria  retained  their  at- 
tenuation. According  to  Chamberland  and  Roux, 
the  test  for  the  attenuation  of  the  virulence  according 
to  any  method,  should  be  the  absence  of  spurules  in 
the  filaments. 

Koch,  Gaffky,  and  Loefflers  Method. — These  ex- 
perimenters used  the  thermostat  of  D'Arsonval,  which 
admits  of  a  variation  of  temperature  of  only  one-tenth 
to  one  degree.  They  also  used  the  culture  flask  of 
Erlenmeyer.  Each  flask  containing  20  c.c.  of  chicken- 
broth,  neutralized  by.  carbonate  of  soda.  After  the 
flasks  had  been  inoculated  they  were  placed  in  the 
thermostat  at  42°  to  43°  C.,  and  the  degree  of  attenu- 
ation was  found  by  experiments  on  the  lower  animals, 
such  as  mice,  rabbits,  and  the  like.  They  came  to 
the  conclusion  that  methods  for  the  protective  inocu- 
lation which  had  hitherto  been  recommended,  were  of 
doubtful  advantage. 

How  to  Prepare  the  Bacilli  of  Anthrax. — The 
bacilli  may  be  stained  with  ordinary  aniline  dyes,  and 
in  various  ways.  It  is  best,  however,  to  decolorize  the 
tissues  if  they  are  in  solid  organs,  and  Gram's  rm  thod 
is  to  be  recommended  for  this  purpose.  Weigert's 
double  staining  method  is  also  recommended. 


—  66  - 

I  will  now  say  a  few  words  about  the  morphology 
of  the  microzymes  found  in  infected 

CHOLERA. 

The  comma  bacillus,  which  Koch  claims  to  be 
pathognomonic  of  cholera,  is  a  slender  body  with  a 
rounded  extremity  blended  with  the  former.  Much 
dispute  has  arisen  in  reference  to  this  bacillus,  Lewis, 
of  India,  claiming  that  a  similar  bacillus  can  be  found 
in  the  secretion  of  the  human  mouth,  while  Finckler 
and  Pryor  claim  that  a  similar  comma  bacillus  may 
be  found  in  the  dejections  of  cholera  nostras  patients. 
But  Koch  has  claimed,  in  reply  to  these  last  objec- 
tions, that  the  two  comma  bacilli  have  a  different 
growth  in  gelatine  or  potato,  and  that  there  are  other 
morphological  differences. 

Koch's  Method  iw  making  a  diagnosis  in  epidemic 
cholera  is  as  follows:*  The  intestines  of  Asiatic 
cholera  patients  show  an  infiltration  with  bacteria 
which  are  partly  in  the  glands,  and  partly  between  the 
epithelium  and  the  basement  membrane.  The  intes- 
tinal contents,  however,  exhibit  great  variety  in  the 
form  of  bacteria.  The  bacillus  of  cholera  is  claimed 
to  be  smaller  than  that  of  tuberculosis,  being  about 
two  thirds  the  length,  but  it  is  firmer  and  thicker  than 
the  latter,  and  has  a  slightly  bent  appearance.  Some- 
times the  growth  is  doubled  so  that  the  bacillus  is 
shaped  like  the  letter  S.  This  peculiar  conformation 

*  Koch's  Report  at  the  Cholera  Convention,  July  26,  '84. 


-67  - 

is  due  to  the  union  of  two  individuals.  In  pure  cul- 
tures another  form  is  recognized.  This  consists  of 
rather  long  filaments  similar  to  the  spirilla  of  recurrent 
fever;  so  very  similar  are  they  that  Koch  thinks  he 
would  be  unable  to  distinguish  the  two.  Cultivated 
in  meat  broths  the  cholera  bacilli  increase  with  great 
rapidity,  and  are  unusually  active.  They  also  grow 
abundantly  and  rapidly  in  milk,  but  do  not  cause  it  to 
curd1^;  it  appears  unchanged,  but  if  a  small  drop  be 
taken  from  the  surface  and  examined  microscopically, 
it  will  be  found  to  teem  with  cholera  bacilli.  The 
cholera  bacilli  also  grow  well  in  blood-serum,  and  also 
in  peptone-gelatin.  The  comma  bacillus  may  be 
cultivated  upon  agar-agar.  Cultures  may  also  be 
made  upon  boiled  potato  when  they  develop  growths 
like  those  of  the  bacilli  of  glanders.  It  seems  that  the 
vegetation  of  the  bacilli  in  gelatine  not  only  dissolves 
the  latter,  but  thins  the  fluid  and  in  so  doing  they 
form  cup-like  cavities  on  the  surface;  these  peculiari- 
ties are  supposed  to  distinguish  comma  bacilli  from 
other  bacilli.  The  comma  bacilli  germinate  best  at  a 
temperature  between  30°  and  40°  C.;  below  17°  the 
development  is  slow,  and  ceases  at  16°.  Koch  believes 
that  if  the  cholera  bacilli  are  introduced  from  the  in- 
testine into  a  putrid  fluid  containing  the  productions 
of  the  decomposition  of  other  bacteria,  they  will  not 
develop  well,  but  will  soon  die. 

Nicati's    and   RietscWs    Method. — They  took    a 
Pasteur  filter  and   pass  an  eight-day  pure  culture  in 


—  68  — 

broth  or  gelatin  through  it.  The  filtrate  was  injected 
into  the  veins  of  a  dog,  which  afterwards  exhibited  all 
the  symptoms  of  cholera. 

It  is  interesting  to  note  here  that  Rochefontaine 
has  swallowed  the  alvine  dejections  of  a  cholera  pa- 
tient made  into  five  large,  soft  pills.  These  pills  he 
took  successfully  with  water.  The  pulse  rose  to  100, 
the  skin  became  hot,  he  had  slight  nausea,  dysuria, 
slight  convulsion  of  the  muscles  of  the  legs,  etc.,  and 
constipation  for  twenty-four  hours.  He  then  took  a 
glass  of  alkaline  water  and  felt  well  again.* 

GLANDERS. 

The  bacterium  associated  with  this  disease  has 
been  called  the  bacillus  malandriae. 

Loeffler's  and  Schutz's  Method. — A  solid  culture  is 
made  from  the  blood-serum  of  a  horse.  This  is  then 
inoculated  with  the  matter  from  a  fresh  tubercle. 
After  an  interval  of  three  days  the  surface  exhibits  a 
number  of  small  transparent  spaces;  in  these  there  are 
small  bacilli  similar  to  those  that  occur  in  the  lung, 
liver,  spleen,  etc.  In  staining  these  bodies,  methyl- 
blue  is  used  in  a  concentrated  solution. 

HOG    CHOLERA. 

Synonym:     Swine-plague. 

Opinions  differ  as  to  the  form  of  the  bacterium 


*  Comptes  Rend.  Tom.  99,  Nov.  17,  1884,  p.  845. 


_69- 

associated  with  this  disease.  Klein*  holds  that  it  is  a 
bacillus  of  very  small  size;  others  that  it  is  a  micro- 
coccus  of  the  link  form,  called  diplo-coccus.  The 
last  named  form  has  been  described  by  Pasteur,  f 

HYDROPHOBIA. 

Pasteur  has  described  in  this  disease  a  bacillus 
lyssae.  As  in  the  former  case,  it  is  still  a  matter  of 
dispute  whether  the  micro-organism  associated  with 
the  disease  is  a  bacillus  or  a  micrococcus.  Pasteur 
adheres  to  the  former  view;  others  maintain  the  latter 
one. 

Pasteur  s  Method  of  Inoculation. — The  animal  is 
trephined  and  the  virus  is  injected,  with  an  admixture 
of  water,  into  the  membranes  of  the  brain,  by  a  hypo- 
dermic syringe.  He  states  that  in  fifteen  to  twenty 
days  the  animals  die  of  rabies,  and  the  diseased  brain 
is  then  capable  of  producing  hydrophobia  in  other 
animals.  His  statements  are  as  follows: 

First.  If  the  virus  of  rabies  is  transferred  from  a 
dog  to  a  monkey,  and  then  to  other  monkeys,  it  grad- 
ually becomes  weaker;  and,  if  it  be  injected  in  this 
state  into  a  dog,  rabbit,  or  guinea  pig,  it  remains  in 
the  same  condition. 

Second.     On   the   other   hand,  the  poison  is  in- 


*  The  Report  of  Infectious  Pneumo-enteritis  of  the  Med- 
ical Office  of  the  Privy  Council,  1877-78,  London. 

f  Pasteur  and  Thuillier,  Vacc.  de  Rouges  de  Pore.  Comp. 
Rend.,  Tom.  97,  p.  1163. 


creased  if  it  be  transferred  from  one  rabbit  to  another, 
or  from  one  guinea-pig  to  another.  Now,  in  this  in- 
tensified condition,  the  virus  is  inoculated  upon  a  dog, 
in  which  case  it  invariably  produces  death.  But,  al- 
though the  virulence  of  the  poison  may  be  thus  in- 
creased by  a  transference  from  one  rabbit  to  another, 
it  is  necessary  to  repeat  the  process  several  times  if  it 
has  been  attenuated  by  inoculation  upon  a  monkey; 
and  hence,  Pasteur  claims  that  he  can  render  the  ani- 
mal insusceptible  to  the  real  disease.  If,  for  example, 
it  be  desirable  to  make  a  dog  refractory  to  the  real 
disease,  the  virus  is  first  inoculated  upon  several  rab- 
bits, but  inoculated  upon  the  dog  at  every  successive 
inoculation  upon  a  rabbit.* 

Gibiers  Method  of  Attenuation. — He  exposes  the 
virus  of  rabies  to  a  very  low  temperature  in  order  to 
weaken  it.  His  method  is  as  follows: 

He  takes  a  small  drill  and  perforates  the  skull  in 
the  median  line;  the  virus  is  then  injected  by  means 
of  a  hypodermic  syringe.  The  value  of  this  mjethod 
has  not  been  satisfactorily  determined. 

Babes'  Method  for  Staining. — He  simply  makes 
cover-glass  preparations  of  the  saliva,  using  the  ordin- 
ary methyl-violet  fluid;  aniline-red  fluids  are  also 
used.  It  is  improbab'e  that  the  bacteria  in  these  cases 
have  been  successfully  isolated. 


*  Pasteur,  Comp.  Rend.  Tom.  92,  p.  1260. 


_  7I  __ 

Koch*  contends  that  the  organisms  which  Pasteurf 
has  described  were  simply  those  that  belong  to  septi- 
caemia. 

LEPROSY. 

The  bacterium  of  this  disease  has  been  called  the 
bacillus  leprae  by  Hansen.J  The  bacilli  are  said  to  be 
organisms  which  do  not  give  up  in  acid  solutions 
the  staining  or  color  which  they  have  previously  taken. 
Bismarck-brown  will  not  stain  them,  but  the  blue, 
violet,  and  red  aniline  colors  will. 

Baumgartens  Method, — He  takes  a  saturated 
alcoholic  solution  of  aniline-red  and  adds  five  drops  of 
it  to  a  watch-glass  of  distilled  water.  The  cover- 
glasses  are  floated  upon  this  liquid  for  six  or  seven 
minutes,  with  the  bacilli  upon  them.  They  are  then 
placed  for  fifteen  seconds  in  acidulated  alcohol.  To 
decolorize  the  preparations,  use  the  following  prepara- 
tion: Nitric  acid,  one  part;  alcohol,  two  parts.  They 
are  then  again  stained  with  a  watery  solution  of 
methyl-blue,  subsequently  washed  in  water,  saturated 
with  absolute  alcohol,  clarified  in  oil  of  bergamot,  and 
mounted  in  Canada  balsam.  The  objects  are  then 
studied  with  an  oil  immersion  lens.  It  is  said  that 
tubercular  bacilli  do  not  stain  by  this  method.  When 
staining  the  bacilli  in  sections,  the  latter  should  remain 

*  Ueber  die  Milz  brand  Impfung,  Berlin,  1882,  p.  5. 
f  Recherch.  Sous  la  Rage,  Comp.  Rend.,  Tom.  98,  1884. 
Et  Pasteur  Comp.  Rend.,  Tom.  98,  No.  8,  p   477,  1884. 
\  Virchow.  Archiv.,  Band  79,  1880. 


—  72  — 

somewhat  longer — twelve  to  fifteen  minutes — in  the 
staining  fluid.  They  should  be  decolorized  for  thirty 
seconds  in  the  solution,  and  washed  three  or  four 
minutes  in  water,  etc.  '  The  restaining  with  methyl- 
blue  should  occupy  two  or  three  minutes.  The  lepra 
bacilli,  by  'this  method,  assume  after  a  few  minutes  a 
red  color.  The  bacilli  of  tuberculosis  are  colorless  if 
present. 

Neisser's  Method* — Neisser  stains  in  gentian- 
violet  or  methyl-violet.  His  culture  method  consists 
in  taking  a  lepra  tubercle,  making  cultures  from  it 
under  proper  precautions  in  blood-serum,  sterilized, 
etc.  The  preparations  are  kept  in  an  oven  at  from 
35°  to  39°  C. 

BACULLIS    OF    MALARIA    (KLEBS). 

Klebs  and  Tomassi-Crudelli' s  Method. — The  sup- 
posed infective  material  was  taken  from  the  air  of  a 
malarial  district.  The  germs  were  collected  by  forc- 
ing the  air  against  a  glass  plate  standing  at  right 
angles  to  the  air  current.  The  plate  was  covered  with 
glycerine-gelatin.  From  this  plate  fractional  cultures 
were  made  in  fluid  media  of  various  kinds.  After 
passing  these  pure  cultures  through  plaster-of- Paris 
filters,  animals  were  inoculated,  first  with  the  unfiltered 
material,  and  then  with  the  filtrate.  The  latter  ex- 
hibited only  a  slight  rise  of  temperature;  the  former 
had  typical  malarial  fever.  In  order  to  obtain  the 

*  Neisser,  Virchow,  Archiv.,  Band  84,  1884. 


—  73  — 

germs  from  the  earth,  they  took  a  porcelain  vessel  of 
large  size  and  placed  it  upon  a  sand  bath.  The  vessel 
was  then  well  lined  with  damp  earth;  it  was  kept 
moistened  from  time  to  time  with  a  little  water.  Upon 
this  prepared  earth  was  placed  a  metal  box  contain- 
ing the  earth  to  be  examined;  the  bottom  of  it  was 
perforated  with  numerous  openings.  The  whole  ap- 
paratus was  then  kept  at  a  temperature  of  from  30°  to 
35°  C.  Fractional  cultures  were  then  made  from  the 
earth  in  the  box,  and  animals  were  inoculated.  It  was 
found  that  the  germs  would  not  pass  through  the 
plaster- of- Paris  filters.* 

Richard's  Method. — He  makes  a  direct  examina- 
tion of  the  blood  taken  from  a  patient's  finger.  This 
method  is  the  best  to  be  employed  for  studying  the 
microbe  in  its  movements.  In  order  to  see  the  bac- 
teria in  cases  where  there  are  very  few  present,  he 
destroys  the  normal  red  corpuscle  by  adding  to  a 
drop  of  the  blood  a  drop  of  acetic  acid;  the  bacteria 
are  then  readily  brought  into  view.  He  finds  that  the 
microbe  has  a  special  affinity  for  the  red  corpuscle  in 
which  it  is  developed.! 

SYPHILIS. 

In  this  disease  also,  as  in  others  that  have  been 
mentioned,  there  is  a  dispute  as  to  whether  the  special 
bacterium  is  a  micrococcus  or  a  bacillus. 


*  Tomassi-Crudelli,  Archiv  fiir  Exper.  Path,  und  Phar., 
Band  12,  Heft  3,  p.  225. 

f  Comp    Rend.  No.  8,  1882. 


—  74  — 

Birsch-Hirschf eld's  Method. — He  takes  syphilitic 
deposits  or  lesions  that  have  been  hardened  in  abso- 
lute alcohol,  then  stains  them  in  a  concentrated  solu- 
tion of  aniline-red,  washes  in  distilled  water,  and 
mounts  in  the  usual  way.f 

TUBERCULOSIS. 

The  bacillus  tuberculosis  is  the  special  parasite 
which  has  been  connected  with  the  disease  by  Koch. 
Tubercular  bacilli  resemble  the  bacilli  of  leprosy  in  so 
far  as  they  will  retain  their  color  even  after  immersion 
in  strong  acids,  and  this  quality  is  said  to  be  enjoyed 
in  common  with  them  by  the  bacilli  of  leprosy  only,  as 
we  have  seen.  These  bacilli  also  often  exhibit  round 
or  oval  spores  in  their  interior.  The  best  method, 
which  has  been  generally  accepted  for  staining  bacilli, 
is  that  of  Ehrlich. 

Ehrlictis  .Method. — He  takes  a  small  amount  of 
sputum,  presses  it  between  two  cover-glasses,  and 
separating  them,  passes  them,  with  the  surface  turned 
upward,  through  the  gas  flame.  He  then  turns 
them  with  the  preparation  on  the  lower  side  and 
floats  them  in  a  watch-glass  filled  with  gentian-violet, 
methyl-violet,  or  his  fuchsine  aniline  oil  solution,  al- 
lowing them  to  remain  there  for  from  fifteen  minutes 
to  half  an  hour.  He  then  heats  the  whole  over  a 
flame  until  it  steams  for  one  minute,  then  washes  in  a 

f  Birsch-Hirschfeld,  Centralbl.  fur  die  Med.  Wissen., 
1882  No.  33. 


—  75  — 

solution  of  nitric  acid,  33  per  cent.,  so  that  the  color 
fades  out  of  the  matrix,  the  bacilli  alone  retaining  it, 
then  washes  in  distilled  water,  dries,  passes  through 
absolute  alcohol,  and  mounts  in  balsam.  In  order  to 
make  the  picture  clearer  he  then  restains  the  decolor- 
ized matrix.  This  is  best  done  by  some  contrasting 
color,  such  as  Bismarck-brown, vesuvine,  or  the  like. 

To  Stain  Sections.  —Sections  should  remain  in  the 
staining  fluid  for  twenty-four  hours,  and  for  two  or 
three  minutes  in  the  acid  mixture.  Wash  well  in  water 
several  times,  and  then  mount  in  the  ordinary  way. 

Ziehl's  Method. — In  this  method  carbolic  acid  is 
substituted  for  aniline  oil  and  caustic  potash.  This 
method  consists  in  preparing,  as  in  Ehrlich's  method, 
but  omitting  the  decolorization,  by  the  nitric  acid  solu- 
tion. In  place  of  aniline  oil  he  uses  pyrogallic  acid 
and  resorcin,  and  he  was  the  first  to  show  that  the 
tubercular  bacillus  does  not  need  an  alkaline  staining 
fluid.  Still  later,  Dr.  Pryor  showed  that  Ehrlich's 
method  was  not  alkaline,  but  neutral,  and  that  oil  of 
turpentine  might  take  the  place  of  the  aniline  oil. 

Among  other  methods  are  those  of  Fraenzel*  and 
of  Rindfleisch. 

Baumgartens  Culture  Method. — Baumgarten  took 
a  small  fragment  from  a  human  nodule  and  intro- 
duced it  with  antiseptic  precautions  into  the  anterior 
chamber  of  the  eye  of  the  living  rabbit.  Here  it  in- 
creased in  size.  He  then  removed  this  particle  and 

*  Berliner  Klin.  Wochensch.,  1882,  No.  145. 


-  76  - 

placed  it  into  the  anterior  chamber  of  the  eye  of  an- 
other rabbit,  and  in  six  or  eight  days  in  a  third  rabbit. 
This  method  requires  no  trouble  and  needs  no  special 
apparatus,  and  a  pure  culture  is  thus  obtained  outside 
of  the  living  body. 

Reinstadlers  Culture  Method. — He  took  a  piece  of 
tubercular  lung,  placed  it  in  a  sterilized  vessel  with 
some  sand  that  had  been  heated  red  hot.  To  this 
material  he  added  a  quantity  of  Bergman's  fluid,  which 
had  been  thoroughly  super-heated.  He  filtered  this 
liquid  and  sealed  for  use.  Some  test-tubes  were  then 
cleansed  by  boiling  in  nitric  and  sulphuric  acid,  and 
then  in  alcohol;  they  were  then  super-heated  in  a 
spirit  flame  and  plugged  with  carbolized  cotton;  then 
they  were  filled  with  a  sterilized  pipette  with  30  c.  c. 
of  Bergman's  fluid,  and  the  whole  is  sterilized  by  heat, 
and  cooled. 

Celli  and  Guarnieri's  Method. — To  examine  the 
air  used  by  consumptive  patients,  they  took  a  large 
tin  cylinder,  bent  at  the  top,  and  terminating  below  in 
a  conical  extremity  whose  apex  contained  a  small 
pointed  tube,  the  free  end  of  which  terminated  in  a 
wide  cone  of  tin,  into  which  was  introduced  a  cone  of 
copper  bent  at  its  apex,  and  the  surface  of  which  was 
coated  with  Koch's  gelatine.  The  gas  flame  caused  a 
current  of  air  to  pass  over  the  gelatin  surface;  a  gas 
jet  was  placed  in  the  lower  part  of  the  large  tin 
cylinder.  The  gelatine  was  kept  at  an  even  tempera- 
ture of  30°  to  40°  C.  Examinations  were  made  in  the 


—  77  ~ 

usual  way.  They  also  made  further  experiments  by 
requiring  patients  to  breathe  repeatedly  for  twenty- 
four  hours  upon  a  wooden  dish  coated  with  Koch's 
gelatine,  and  covered  by  a  watch-glass.  They  also 
used  a  variety  of  other  methods  and  experimented 
with  sputum.  They  found  that  the  sputum,  so  long 
as  it  is  moist,  does  not  evolve  any  specific  bacilli. 


FIG.  19. 

Hesse's  apparatus  for  examining  ordinary  atmos- 
pheric or  contaminated  air  consists  of  a  hollow  glass 


cylinder,  18  inches  in  length  and  2^/2,  in  diameter. 
One  end  of  the  tube  is  fitted  with  a  rubber  cap  per- 
forated by  a  minute  orifice.  The  other  end  is  occupied 
by  a  perforated  India-rubber  cork,  pierced  by  a  short 
piece  of  glass  tubing.  To  the  tubing  is  attached  a 
litre  flask,  that  is  again  similarly  connected  with 
another  litre  flask.  The  cylinder  is  sterilized  by 
superheating  in  the  steam  apparatus,  or  by  washing 
with  a  y^o-  solution  of  the  bichloride  of  mercury. 
Then  a  thin  layer  of  nutrient  gelatine  is  introduced 
into  the  tube,  so  as  to  make  a  thin  coating  along  the 
floor.  After  shutting  off  the  external  air  by  clamping 
the  rubber  tube  at  one  end  and  putting  a  cap  of  cotton 
in  the  other  the  apparatus  is  thoroughly  sterilized  in 
the  ordinary  way.  After  cooling,  the  gelatine  will 
solidify.  Next,  the  litre  flasks  having  been  filled  with 
water  and  the  clamp  and  cotton  cap  removed,  the 
flasks  are  allowed  to  siphon  themselves  off  drop  by 
drop.  In  so  doing  the  atmospheric  air  enters  through 
the  minute  opening  in  the  rubber  cap  and  the  atmos- 
pheric germs  also  enter  and  fall  by  gravitation  upon 
the  nutrient  medium,  where  they  are  permitted  to 
develop.  A  variety  of  other  methods  for  collecting 
atmospheric  germs  are  also  employed,  many  of  them 
being  of  an  extremely  simple  character. 

TYPHOID    FEVER. 

The  bacilli  of  typhoid  fever  were  first  named  by 
Brautlecht.*     If  Gram's  method  is  used  the  bacilli  are 

*  Virchow,  Archiv.,  1880,  p.  80. 


—  79  — 

decolorized.  In  this  case,  as  in  the  foregoing,  there 
is  a  difference  of  opinion  as  to  whether  the  bacterium 
is  a  micrococcos  or  a  bacillus. 

LetzericKs  Method. — His  plan  was  to  take  fresh 
tissues,  or  those  that  were  partly  hardened,  make  sec- 
tions and  clarify  them  with  weak  solutions  of  caustic 
potash  or  carbonate  of  soda,  or  even  in  diluted  acetic 
acid.  *  He  mounted  them  in  glycerine  and  water. 

Rindfleisctts  Method. — He  took  water  from  a 
suspected  well,  placed  a  drop  upon  a  slide,  colored  it 
with  a  solution  of  methyl-violet,  washed  in  water, 
dried,  and  mounted  in  Canada  balsam.  Some  deep 
blue  rod  bacteria  were  brought  into  view.  He  then 
inoculated  with  the  water  gelatin  made  of  human 
flesh,  under  proper  precautions.  The  gelatin  was 
rapidly  dissolved  by  the  culture.* 

CHICKEN    CHOLERA. 

Klein  believes  that  the  organism  Pasteur  has  de- 
scribed is  a  bacterium  termo  of  putrefaction.  Others 
call  it  a  micrococcus  or  diplo-coccus.  For  a  culture 
fluid  in  this  case  Pasteur  employed  a  meat  or  chicken 
broth,  neutralized  with  carbonate  of  potash,  and 
sterilized  at  a  temperature  of  100°  to  115°  C.  After 
cultivating  these  microbes  for  a  few  days,  he  inocu- 
lated a  chicken,  producing  the  disease  in  a  mild  form. 


*  Sitz.    Bericht  der  Phys.   Med.   Gesell.  zu  Wurz,   1882, 
P.  133- 


—  8o  — 

DIPHTHERIA. 

The  organism  in  this  case  has  been  called  the 
micrococcus  diphtheriticus  by  Cohn. 

The  same  difference  of  opinion  occurs  among  in- 
vestigators as  to  whether  the  organism  in  this  case  is 
a  spherical-  or  a  rod-shaped  body.  Klein  and  others 
believe  that  the  spherical  bodies  are  the  germs  of  the 
disease. 

Loeffler  s  Method. — He  takes  30  c.  c.  of  concen- 
trated alcoholic  methyl-blue  solution,  TOO  c.  c.  of 
caustic  potash  solution,  and  in  the  proportion  of  one 
to  1000  of  water.  The  sections  remain  only  for  a 
few  moments  in  this  solution,  which  gives  a  deep  stain 
to  all  known  bacteria.  The  sections  are  then  placed 
for  a  short  time  in  one-half-per-cent.  acetic  acid  solu- 
tion in  the  ordinary  way,  and  mounted  according  to 
ordinary  rules.  This  method  demonstrated  to  Loeffler 
two  forms;  one,  the  chain  bacterium,  which  he  was 
able  to  cultivate  in  blood-serum  and  on  cooked  po- 
tato; and  the  other,  a  bacillus  that  grew  at  37°  C.,  in 
a  mixture  of  three  parts  of  calves'  or  sheep's  serum  to 
one  of  neutralized  beef  broth,  one  per  cent,  peptone, 
one  per  cent,  beet  sugar,  and  i  per  cent.  salt.  These 
bacilli  were  regarded  by  Loeffler  as  active  promoters 
of  the  disease,  while  the  spherical  forms  he  thought 
were  unimportant. 

ERYSIPELAS. 

Fehleiserfs  Method. — The  material  he  takes  from 


—  8i  — 

an  erysipelatous  blister,  together  with  a  part  of  the 
skin,  washes  it  with  ether,  and  sprays  it  with  a  sublim- 
ate solution.  The  diseased  tissue  is  then  put  in  nutri- 
ent gelatine  or  blood-serum,  and  is  cultivated  in  the 
ordinary  way.  H'aving  obtained  a  culture,  he  inocu- 
lated a  woman  58  years  old,  who  was  dying  with 
multiple  sarcomata  of  the  skin.  A  typical  case  of 
erysipelas  resulted.  He  found,  as  he  thought,  that 
these  inoculations  in  other  cases  favorably  influenced 
the  progress  of  neoplasms.  This  was  true  in  a  case 
of  cancer,  another  of  lupus,  and  another  of  sarcoma. 
A  three-per-cent.  solution  of  carbolic  acid  destroys 
the  poison.* 

GONORRHOEA. 

Neissers  Method. —  To  stain  the  micrococcus 
gonorrhcese,  he  spreads  the  secretion  thinly  upon  a 
sterilized  coverglass.  This  is  best  done  by  plac;rg  a 
drop  between  two  glasses,  after  which  they  are  drawn 
apart.  The  preparation  is  dried  in  the  air,  heated  up 
to  120°  C.,  and  allowed  to  remain  at  that  temperature 
for  one  or  two  hours.  The  specimen  should  be  well 
stained.  There  is  no  staining  fluid  so  good  as  methyl- 
blue,  which  stains  the  bodies  rapidly.  The  cover- 
glasses  remain  in  the  dye  from  twelve  to  twenty-four 
hours.  They  are  best  studied  with  a  high  immersion 
power. 

*  Fehleisen,  Sitz.  Bericht,  Med.  Phys.  Gesell.,  Wurzburg, 
1883,  No.  i. 

6  L 


—    82    — 

To  produce  a  culture,  prepare  neutralized  beef 
extract  gelatine,  or  neutralized  blood-serum,  etc.  As 
for  inoculation,  it  has  been  shown  that  dogs  have  im- 
munity, also  rabbits;  but  when  the  pure  culture  was 
injected  into  the  urethra  of  a  man  46  years  old,  he 
got  up  a  true  gonorrhoea.* 

CROUPOUS    PNEUMONIA. 

Friedlaender  s  Method.\ — In  order  to  make  a  cul- 
ture, Friedlaender  takes  portions  of  lung  from  a  patient 
who  has  died  from  the  disease,  removing  them  with  a 
sterilized  wire,  and  cultivates  in  blood-serum  according 
to  Koch's  plan.  The  pure  cultures  have  a  character- 
istic nail  shape  in  gelatine.  The  micrococci  are 
characterized  in  may  cases  by  a  peculiar  capsule.  It 
is  thought  by  some  that  this  is  due  to  the  mode  of 
preparation  and  staining.  In  staining  tissues  the  sec- 
tions should  remain  for  one  hour  in  the  aniline  gen- 
tian-violet solution  at  45°  C.  They  should  then  be 
washed  in  the  iodo-iodide  of  potash  solution,  consist- 
ing of  iodine,  i  part;  iodide  of  potassium,  2  parts;  and 
water,  300  parts;  passed  through  absolute  alcohol  and 
oil  of  cloves,  and  mounted  in  Canada  balsam. 

RECURRENT    FEVER. 

The  spirilla  are  different  from  all  other  bacteria, 
in  so  far  as  they  are  destroyed  by  acids,  alkalies,  and 


*  Neisser,  Deutsche  Med.  Wochenblatt,  1884,  p.  279. 
f  Med.  Bericht,  Clin.  Wochenschrift,  1883,  No.  48,  p.  752. 


-  83  - 

even  by  distilled  water.  No  one  has  succeeded  in 
staining  them  but  Koch,  who  used  brown  aniline,  and 
succeeded  in  photographing  them. 

Friedlaender 's  Method* — His  method  of  obtain- 
ing these  bodies  is  to  take  blood  from  a  patient  by 
means  of  a  cupping-glass  and  allow  it  to  coagulate; 
the  spirilla  remain  on  the  surface  of  the  clot  and  retain 
their  characteristics  for  days.  They  occur  in  the 
blood  only  during  the  febrile  attacks. 

YELLOW    FEVER. 

Friere's  Method. — This  was  to  cultivate  the  organ- 
isms obtained  from  the  blood  by  general  methods,  and 
to  attenuate  the  cultures  by  heat  in  the  manner  em- 
ployed by  Pasteur  for  anthrax.  In  1883  he  received 
permission  from  the  emperor  of  Brazil  to  inoculate 
some  of  his  subjects.  He  operated  several  hundred 
times,  and  is  said  to  have  produced  the  disease  in  a 
mild  form.  The  immunity  conferred  seemed  to  be 
perfect*. 

VARIOLA. 

The  micrococcus  variolse  et  vaccinse,  according  to 
the  name  given  by  Cohnf ,  is  the  cause  of  the  disease, 


*  Koch,   Deut.   Med.  Wissen.,  No.  19,  1879;    Obermeier, 
Berlin.  Klin.  Wochenbl.,  1873,  pp.  152  und  391. 

*  Friere  et  Rabougeon,   Comp.   Rend.  Acad.  de  Science 
de  Paris,  Tom.  99,  Nov.  10,  1884. 

f  Virchow  Archiv.,  1872. 


—  84- 

but  as  yet  no  successful  cultures  or  inoculations  have 
been  performed.  But,  in  view  of  successful  experi- 
ments, an  English  Company,  the  Worshipful  Company 
of  Grocers,  offered  a  prize  of  five  thousand  dollars  to 
the  discoverer  of  a  method  by  which  the  vaccine  con- 
tagion may  be  cultivated  apart  from  the  animal  body. 
The  prize  was  open  to  universal  competition,  British 
and  foreign,  and  the  paper  was  to  be  submitted  before 
December  31,  1886.  The  committee  who  had  the 
matter  in  charge  were  John  Simon,  Burdon  Sanderson, 
George  Buchanan,  and  others.  Their  report  has  not 
yet  been  read. 

In  concluding  this  brief  summary,  it  will  be  ob- 
served that  of  the  sixteen  diseases  enumerated,  in  at 
least  eight  the  form  of  the  microphyte  is  in  dispute. 
It  is  safe  to  say,  however,  that  there  is  even  less  cer- 
tainty in  the  matter  than  this  ratio  indicates. 


Finely  mounted  and  stained  preparations  of  the 
following  forms  of  bacteria  and  fungi,  pathogenic  and 
innocent,  at  85  cents  per  slide,  or  $9.00  per  dozen,  can 
be  obtained  from  J.  W.  Queen  &  Co.,  924  Chestnut 
St.,  Philadelphia: 

Bacillus  tuberculosis. — $1.00. 

"       subtilis  (innocent  bacillus). 
"        of  anthrax  (sheep). 
"       of  sour  milk. 
"       of  vinegar. 


-  85  - 

Micrococcus  pneumonicus. 
"  diphtheriticus. 

"  gonorrhoeicus. 

"  of  urine. 

"  prodigiosus. 

•  "  of  vaccine  virus. 

Spirochaete  biiccalis  (mouth). 

"  obermeieri  (from  typhus  recurrens). 

Saccharomyces  cerevisii  (upper  yeast). 
"  "         (lower  yeast). 

(These  two  are  unstained;  price,  75  cts.  each.) 
Satcina  ventriculi,  from  stomach. 
Oidium  albicans,  from  mouth. 
Achorion  schoenleini  (favus). 
T.  H.  McAllister,   manufacturing  optician,  of  49 
Nassau   St.,   New   York  City,   has  a  still  larger  and 
almost  complete  series  of  the  stained  and  unstained 
bacteria  found  in  the  several  infective  diseases. 


RETURN  TO  the  circulation  desk  of  any 
University  of  California  Library 
or  to  the 

NORTHERN  REGIONAL  LIBRARY  FACILITY 
Bldg.  400,  Richmond  Field  Station 
University  of  California 
Richmond,  CA  94804-4698 

ALL  BOOKS  MAY  BE  RECALLED  AFTER  7  DAYS 
2-month  loans  may  be  renewed  by  calling 

(415)642-6233 
1-year  loans  may  be  recharged  by  bringing  books 

to  NRLF 
Renewals  and  recharges  may  be  made  4  days 

prior  to  due  date 

DUE  AS  STAMPED  BELOW 


LIBRARY  USE  JAN  15 '87 


YC  88549 


Micr.  Soc 


9/ 


OLOGY 


THE  UNIVERSITY  OF  CALIFORNIA  LIBRARY 


